U.S. patent application number 11/987217 was filed with the patent office on 2008-06-26 for procedure for the optical acquisition of objects by means of a light raster microscope with line by line scanning.
Invention is credited to Ralf Engelmann, Frank Hecht, Ralf Wolleschensky.
Application Number | 20080149818 11/987217 |
Document ID | / |
Family ID | 34833302 |
Filed Date | 2008-06-26 |
United States Patent
Application |
20080149818 |
Kind Code |
A1 |
Wolleschensky; Ralf ; et
al. |
June 26, 2008 |
Procedure for the optical acquisition of objects by means of a
light raster microscope with line by line scanning
Abstract
Procedure for the image acquisition of objects by means of a
light raster microscope with line by line scanning, whereas a
scanning of the specimen for the creation of a specimen image
occurs in scanning steps and the distance between at least two
scanning steps is variably adjustable and at least a second
scanning of the specimen occurs, during which the position of the
scanning steps is shifted with regard to the scanning direction,
whereas preferably a line by line scanning of the specimen is
carried out.
Inventors: |
Wolleschensky; Ralf;
(Apolda, DE) ; Hecht; Frank; (Welmar, DE) ;
Engelmann; Ralf; (Jena, DE) |
Correspondence
Address: |
JACOBSON HOLMAN PLLC
400 SEVENTH STREET N.W., SUITE 600
WASHINGTON
DC
20004
US
|
Family ID: |
34833302 |
Appl. No.: |
11/987217 |
Filed: |
November 28, 2007 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
10967326 |
Oct 19, 2004 |
7323679 |
|
|
11987217 |
|
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Current U.S.
Class: |
250/234 |
Current CPC
Class: |
G02B 21/0048
20130101 |
Class at
Publication: |
250/234 |
International
Class: |
H01J 3/14 20060101
H01J003/14 |
Foreign Application Data
Date |
Code |
Application Number |
Jul 16, 2004 |
DE |
10 2004 034 951.7 |
Claims
1. Process for image acquisition of objects using a light raster
microscope with line by line scanning, comprising the steps of: (a)
scanning of a specimen line by line in parallel scan lines in a
scanning direction for the creation of a specimen image, wherein
the distance between at least two scan lines is variably
adjustable, and (b) carrying out at least a second scanning of the
specimen, during which the position of the scan lines is shifted
with regard to the scanning direction.
2. Process according to claim 1, further comprising the step of
changing the proportion between spatial and temporal resolution of
the microscope.
3. Process for studying development processes, using the process of
claim 1, wherein in step (a), the specimen exhibits dynamic
processes on the level of united cell structures and whole
organisms lasting from tenths of seconds up to hours.
4. Process according to claim 3, wherein the specimen is living
cells in a three-dimensional tissue group with markers, which
exhibit manipulation related changes in color by laser
illumination, in combination with living cells in a
three-dimensional tissue group with very weak markers, and wherein
the process further comprises the step of restricting confocality
in favor of detection sensitivity.
5. Process for studying intracellular transportation processes,
using the process of claim 1, wherein the specimen is motile
structures, with high speed in the area of hundredth of seconds in
and wherein the scanning of steps (a) and (b) is carried out using
FRAP with region of interest bleaches.
6. Process for studying molecular and other sub-cellular
interactions, using the process of claim 1, wherein the specimen is
a very small structure with high velocity, and wherein the scanning
of steps (a) and (b) is carried out using indirect techniques with
region of interest bleaches for the resolution of submolecular
structures.
7. Process for studying fast signal transmitting procedures, using
the process of claim 1, wherein the specimen exhibits
neurophysiological processes with high temporal resolution.
Description
CROSS REFERENCE TO RELATED APPLICATIONS
[0001] This application is a continuation application of U.S. Ser.
No. 10/967,326 filed Oct. 19, 2004.
[0002] This application is related to U.S. patent application Ser.
No. 11/716,678 filed Mar. 12, 2007.
BRIEF DESCRIPTION OF THE DRAWINGS
[0003] FIG. 1 is a schematic view of a first embodiment of a laser
scanning microscope in accordance with the present invention.
[0004] FIG. 2 is a diagram showing how a region of interest ROI can
be selected within the available maximum scan field using a zoom
optic.
[0005] FIG. 3 is a schematic view of a second embodiment of a laser
scanning microscope in which a Nipkow disc is used.
[0006] FIG. 4 is a schematic view of a third embodiment of a laser
scanning microscope in which a multipoint scan is used.
[0007] FIG. 5a shows a scan field of a line scanner having parallel
scan lines offset from each other.
[0008] FIG. 5b shows a scan field in which the scan lines are
vertically shifted by a/2 or a/N, N=2, 3.
[0009] FIG. 6 is a schematic illustration of a slider to adjust the
proportion of the spatial and temporal resolution of the microscope
and select the speed of the object to be examined.
[0010] FIG. 7a illustrates a monitor image with high optical
resolution.
[0011] FIG. 7b illustrates a monitor image in which the optical
resolution has been lowered according to the invention (lower image
rate).
[0012] FIG. 8 illustrations the creation of images with a reduced
image rate.
[0013] FIG. 9 shows the connection between the detector resolution
and the number of shifts n.
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
[0014] The invention is explained in detail in the following with
reference to the drawings.
[0015] FIG. 1 shows a scheme of a laser scanning microscope 1,
which basically consists of five components: a radiation source
model 2, which produces the stimulating radiation for the laser
scanning microscopy, a scan module 3, which conditions the
stimulating radiation and diffracts over a specimen for scanning, a
simplified scheme of microscope module 4 directing the scanning
radiation, which is produced by the scan module, in a microscopic
optical path towards a specimen, as well as detector module 5,
which receives and detects optical radiation from the specimen. As
may be seen in FIG. 1, the detector module 5 may be designed with
several spectral channels.
[0016] As a general description of a laser scanning microscope with
point-wise scanning, it is referred to DE 19702753A1, which thus
forms part of the description at hand.
[0017] The radiation source module 2 produces light radiation,
which is suitable for laser scanning microscopy, i.e. particularly
radiation which may trigger fluorescence. Depending on the
application, the radiation source module shows several radiation
sources. In a represented construction, two lasers 6 and 7 are
provided in a radiation source model 2 with a subsequent light
valve 8 and an attenuator 9 which couple their radiation by means
of a coupling 10 into an optical fiber 11. The light valve 8
functions as a baffle, by which a beam deactivation may be achieved
without having to deactivate the operation of the laser in laser
unit 6 or 7. The light valve 8 may for example be designed as AOTF
which deviates the laser beam for beam deactivation before coupling
into the optical fiber 11 towards a light trap, which is not
represented here.
[0018] In the exemplary representation of FIG. 1, laser unit 6
shows three lasers B, C, D, whereas laser unit 7 only includes
laser A. The representation thus serves as an example for a
combined single and multi wave length laser, coupled to one or
several fibers. The coupling may also occur via several fibers at
the same time, the radiation of which is mixed by a color merger
after passing through adaptation optics. It is thus possible to use
various wave lengths or ranges for the stimulating radiation.
[0019] The radiation coupled in optical fiber 11 is combined
through flexible collimate optics 12 and 13 via beam combination
mirrors 14, 15 and changed in a beam forming unit regarding the
beam profile.
[0020] The collimators 12, 13 ensure that the radiation provided to
scan module 3 by radiation source module 2 is collimated in an
indefinite optical path. This preferably occurs with a single
objective which by moving along the optical axis and controlling a
(not represented) central control unit has a focus function, so
that the distance between collimator 12, 13 and the corresponding
end of the optical fiber may be modified.
[0021] The beam forming unit, which will be explained later in
detail, forms from the rotation symmetric Gauss shaped profiled
laser beam, as it occurs after the ray merging mirrors 14, 15, a
linear beam which is no longer rotation symmetric, but rather
creates a rectangular illuminated field in profile.
[0022] This light beam, which is also described as linear, serves
as a stimulating radiation and is led via primary color separator
17 and a zoom objective, which is yet to be described, to a scanner
18. The primary color separator is described later, at this point
it shall simply be said that it separates the specimen radiation,
which returns from the microscope module 4, from the stimulating
radiation.
[0023] The scanner 18 deviates the linear beam uniaxially or
biaxially, after which it is bundled by a scan lens 19 as well as
by a tube lens and a lens of the microscope module 4 into a focus
22, which is located in a compound or specimen. The optical image
is created in a way that the specimen is illuminated in a caustic
line with stimulating radiation.
[0024] This fluorescent radiation stimulated in the linear focus
travels via objective and tube lens of the microscope module 4 and
the scan lens 19 back to the scanner 18, so that in the opposite
direction an inactive beam results from the scanner 18. Therefore,
we also say that the scanner 18 de-scans the fluorescent
radiation.
[0025] The primary color separator 17 lets the fluorescent
radiation pass, which is located in wave length areas other than
the stimulating radiation, so that it may be deviated via a
deviation mirror 24 in the detector module 5 and then analyzed. The
detector module 5 shows several spectral channels in the layout of
FIG. 1, i.e. the fluorescent radiation coming from the deviation
mirror 24 is divided into two spectral channels in an auxiliary
color separator 25.
[0026] Each spectral channel features a slotted diaphragm 26, which
realizes a confocal or partially confocal image in reference to
specimen 23 and the size of which determines the depth of focus
with which the fluorescent radiation may be detected. The geometry
of the slotted diaphragm 26 thus determines the sectional plane
within the (thick) preparation, from which the fluorescent
radiation is detected.
[0027] The slotted diaphragm 26 is followed by a block filter 27
blocking unwanted stimulating radiation which entered the detector
module 5. The linearly expanded radiation, which is separated in
this way, is then analyzed by an appropriate detector 28. The
second spectral detection channel is designed analogously to the
described color channel; it also contains a slotted diaphragm 26a,
a block filter 27a as well as a detector 28a.
[0028] The use of a confocal slotted diaphragm in detector module 5
is only an example. Of course, a single point scanner may also be
used. The slotted diaphragms 26, 26a, are then replaced by
perforated diaphragms and the beam forming unit may then be
eliminated. Otherwise, all optics are designed rotation symmetric
for this model. Instead of a point-wise scanning and detection, any
multi-point configuration such as point clouds or the Nipkow disc
concept may be used, as explained later according to FIGS. 3 and 4.
However, it is then important that the detector 28 achieves local
resolution, since a parallel acquisition of several specimen points
occurs during the scanner's sweep.
[0029] FIG. 1 shows that the Gauss ray beam present according to
the mobile or flexible collimates 12 and 13 is united over mirror
stairs with a ray merging mirror 14, 16 and is subsequently
converted into a ray beam with rectangular ray profile in the model
shown with a slotted diaphragm. In the model of FIG. 1, the beam
forming unit uses a cylinder telescope 37 with a subordinated
aspherical unit 38 and subsequent cylinder optics 39.
[0030] The transformation produces a ray which basically
illuminates a rectangular field in a profile plane, whereas the
intensity distribution along the longitudinal axis of the field is
not Gauss-shaped but rather box-shaped.
[0031] The lighting configuration with the aspheric unit 38 may
serve to evenly fill a pupil between a tube lens and an objective.
In this way, the optical resolution of the objective may be fully
utilized. This option is therefore also appropriate in a single
point or multi point scanning microscope system, e.g. in a line
scanning system (in the latter additionally to the axis in which
the specimen is focused).
[0032] The linear conditioned stimulating radiation is directed
towards the primary color separator 17. It is finished in a
preferred design with a spectrally neutral separator mirror
according to DE 10257237 A1, the content of which is fully included
herein. The term "color separator" thus also includes non-spectral
separating systems. Instead of the described spectrally independent
color separator, a homogenous neutral separator (e.g. 50/50, 70/30,
80/20 or similar.) or a dichroic separator may also be used. To
allow a selection according to the application, the main color
separator is preferably equipped with a mechanism allowing a simple
switch, for example by means of a corresponding separator wheel
containing individual exchangeable separators.
[0033] A dichroic primary color separator is particularly suitable
to detect coherent or directed radiation, such as reflection,
Stokes' or anti-Stokes' Raman spectroscopy, coherent Raman
processes of higher order, generally parametric non-linear optical
processes, such as Second Harmonic Generation, Third Harmonic
Generation, Sum Frequency Generation, dual and multi photon
absorption or fluorescence. Several of these procedures of the
non-linear optical spectroscopy require the use of two or several
laser beams which are collinearly superimposed. Herein, the
represented merging of several laser rays is particularly
beneficial. Basically, the dichroic ray separators common in
fluorescence microscopy may be used. It is also beneficial for
Raman microscopy to use holographic notch separators or filters
prior to the detectors in order to suppress the Rayleigh
distribution fraction.
[0034] In the construction of FIG. 1, the stimulating radiation or
light radiation is fed to scanner 18 via motor controlled zoom
optics 41. Therewith, the zoom factor may be adjusted and the
scanned visual field is continuously variable in a certain
regulating range. Particularly beneficial is a zoom optic where
during the adjustment of the focal position and the image scale,
the pupil position is maintained in the continuous variable
procedure. The three motor degrees of freedom of zoom optic 41,
symbolized in FIG. 1 with arrows, exactly correspond to the number
of degrees of freedom provided for the adjustment of the three
parameters, image scale, focal and pupil position. Particularly
preferable is a zoom optic 41 equipped with a fixed diaphragm 42 on
its departure side. In a practical simple realization, the
diaphragm 42 may also be predetermined by the limitation of the
mirror surface of scanner 18. The diaphragm 42 on the departure
side with zoom optic 41 achieves that, regardless of the adjustment
of the zoom enlargement, a fixed pupil diameter is always projected
onto the scan lens 19. The objective pupil thus remains fully
illuminated in any position of zoom optic 41. The use of an
independent diaphragm 42 prevents the occurrence of any unwanted
scatter in the area of scanner 18.
[0035] Zoom optic 41 cooperates with cylinder telescope 37, which
may also be motor activated and is located in front of the aspheric
unit 38. This option has been chosen in the construction of FIG. 2
to create a compact design; however, it is not mandatory.
[0036] If a zoom factor smaller than 1.0 is required, the cylinder
telescope 37 is automatically pivoted into the optical path. It
prevents an incomplete illumination of the diaphragm 42, when the
zoom lens 41 is reduced. The pivoted cylinder telescope 37 thus
guarantees that even in zoom factors smaller than 1, i.e.
regardless of the adjustment of zoom optic 41, an illumination line
of constant length is always present at the location of the
objective pupil. Compared to a zoom with a simple visual field,
laser performance losses in the light beam may thus be avoided.
[0037] Since in the pivoting of the cylinder telescope 37 an image
brightness shift in the illumination line is inevitable, the (not
represented) control unit is designed in a way that the advance
speed of scanner 18 or an amplification factor of detectors in
detector module 5 is correspondingly adjusted in the activated
cylinder telescope 37, in order to keep the image brightness
steady.
[0038] Apart from the motor driven zoom optic 41 as well as the
motor activated cylinder telescope 37, remote controlled adjusting
elements are also included in the detector module 5 of the laser
scanning microscope of FIG. 1. In order to compensate longitudinal
color errors, for example in front of the slotted diaphragm, a
panorama optic 44 as well as a cylinder optic 39 and immediately in
front of the detector 28, a cylinder optic 39 is included, which
may be motor relocated along the axis.
[0039] In addition to the compensation of errors, a correction unit
40 is included, which will be briefly described below.
[0040] Together with the preceding panorama optic 44, the diaphragm
26 as well as the preceding first cylinder optic 39 and the
following second cylinder optic form a pinhole object of detector
configuration 5, whereas the pinhole is realized here by the
slotted diaphragm 26. In order to prevent unwanted detection of any
stimulating radiation reflecting in the system, the cylinder lens
39 is preceded by a block filter 27, which shows appropriate
spectral characteristics and allows only the desired fluorescent
radiation to reach the detector 28, 28a.
[0041] A modification of the color separator 25 or the block filter
27 inevitably leads to a certain tilt or wedge error at the time of
pivoting. The color separator may lead to an error between the test
area and the slotted diaphragm 26, the block filter 27 to an error
between slotted diaphragm 26 and detector 28. In order to prevent
that a readjustment of the position of the slotted diaphragm 26 or
the detector 28 is required, a plane-parallel disc 40 is located
between the panorama optic 44 and the slotted diaphragm 26, i.e. in
the optical path of the image or the detector 28, which may be
brought into different tilting positions with a controller. The
plane-parallel plate 40 is therefore installed in an appropriate
adjustable holder.
[0042] FIG. 2 shows how with the help of the zoom optic 41a region
of interest ROI may be selected within the available maximum scan
field SF. If the drive of the scanner 18 remains so that the
amplitude does not change, as this may be required in resonance
scanners, an enlargement of more than 1.0 adjusted on the zoom
optic leads to a restriction of the region of interest centered
around the optical axis of the scan field SF.
[0043] Resonance scanners are described for example in Pawley,
Handbook of Biological Confocal Microscopy, Plenum Press 1994, page
461ff.
[0044] If the scanner is controlled in such a way that it scans
asymmetrically to the optical axis or to the idle position of the
scanner mirror, an offset of the region of interest ROI is achieved
in connection with a zoom effect. Through the already indicated
descanning effect of the scanner 18 and by running the zoom optic
41 again, the selection of the region of interest ROI in the
optical path of detection is eliminated again towards the detector.
Any selection within the scan field SF may thus be made for the
region of interest ROI. In addition, images may be received for
different selections of the region of interest ROI and may then
compose those to a high-resolution image.
[0045] If the region of interest ROI shall not only be shifted with
an offset in reference to the optical axis but at the same time
also rotated, a construction is appropriate which provides an
Abbe-Koenig prism in a pupil of the optical path between primary
color separator 17 and specimen 23, which as is known leads to an
image field rotation. This is also eliminated towards the detector.
Now images with various offsets and angles of rotation may be
measured and then combined to a high-resolution image, e.g.
according to an algorithm, as described in a publication by
Gustafsson, M., "Doubling the lateral resolution of wide-field
fluorescence microscopy using structured illumination," in
"Three-dimensional and multidimensional microscopy: Image
acquisition processing VII," Proceedings of SPIE, Vol. 3919 (2000),
p 141-150.
[0046] FIG. 3 shows a further possible construction for a laser
scanning microscope 1, where a Nipkow disc approach is realized.
The light source module 2, which is represented in a very
simplified version in FIG. 3, illuminates a Nipkow disc 64 via mini
lens array 65 through the primary color separator 17, as described
in U.S. Pat. No. 6,028,306, WO 88 07695 or DE 2360197 A1 for
example. The pinholes in the Nipkow disk, which are illuminated
through the mini lens array, are projected onto the specimen in
microscope module 4. In order to be able to vary the image size of
the specimen here as well, a zoom optic 41 is provided.
[0047] In modification of the construction in FIG. 1, in the Nipkow
scanner the illumination occurs through the opening of the primary
color separator 17 and the radiation, which shall be detected, is
reflected. In addition, in modification of FIG. 2, the detector 28
is now designed to achieve local resolution, so that the multipoint
illumination achieved with Nipkow disk 64 is also accordingly
scanned in parallel. Furthermore, an appropriate fixed optic 63
with positive refractive power is arranged between the Nipkow disc
64 and zoom optic 41, which converts the radiation coming through
the pinholes in the Nipkow disc 64 into appropriate bundle
diameters. In the Nipkow construction of FIG. 3, the primary color
separator 17 is a classic dichroic beam separator, i.e. not the
previously mentioned beam separator with a slotted or dotted
reflecting area.
[0048] The zoom optic 41 corresponds to the previously explained
construction, whereas scanner 18 naturally becomes superfluous due
to the Nipkow disc 64. It can still be included if the selection of
a region of interest according to FIG. 2 shall be made. The same
applies for the Abbe-Koenig prism.
[0049] An alternative approach with multipoint scan is shown as a
scheme in FIG. 4, where several light sources irradiate at an angle
into the scanner pupil. Here too, the use of zoom optic 41 for the
projection between primary color separator 17 and scanner 18 allows
the realization of a zoom function as represented in FIG. 2.
Through the simultaneous irradiation of light bundles under
different angles in a plane conjugated to the pupil, light points
in a plane conjugated to the object plane are created which are
directed over a partial area of the entire object plane by scanner
18. The image information results from the evaluation of all
partial images on a matrix detector 28 with local resolution.
[0050] Another version is a multipoint scan, as described in U.S.
Pat. No. 6,028,306, which is fully included here in this regard.
Here too, a detector 28 with local resolution is to be provided.
The specimen is then illuminated through a multipoint light source
which is realized through an irradiation expander with subsequent
micro lens array, which illuminates a multi diaphragm plate in a
way that a multi point light source is realized hereby.
[0051] FIG. 5a shows a scan field of a line scanner with parallel
scan lines SL, which offset from each other.
[0052] Correspondingly, these scan lines may also be created
through line-by-line punctual scanning with a point scanner.
[0053] Offset a is larger here than the distance between the
scanned lines at a scan rate which would lead to a maximally
possible optical resolution of the microscope configuration.
However, an object field may be scanned more rapidly, because the
retention period per recorded line for the image recording
determines the speed of the complete recording.
[0054] In FIG. 5b, the scan lines are vertically shifted by a/2 or
a/N, N=2, 3, the image recording of the individual lines occurs at
distance a, but in the spaces between the scanned lines of the scan
procedure according to FIG. 1a.
[0055] FIG. 6 shows a scheme of a slider to adjust the proportion
of the spatial and temporal resolution of the microscope and select
the speed of the object which shall be examined.
[0056] The scan lines are shifted with the same scan rate at a
certain interval (parallel offset of scanner is changed), but here
not due to the bleaching effect but rather in order to achieve a
compromise in recording rapid processes or movements with a high
demand interval and simultaneous existence of quasi-static or
slowly moving regions or formations in the specimen (almost no
movement), where a low demand interval is required for the
image.
[0057] For example the scan field of 12 mm with 1024 possible lines
is divided into 4 times 256 lines utilizing the optical resolution
and is scanned four times shifted by one line.
[0058] The scanning of 256 lines thus occurs very rapidly. If the
integration time for one line is approx. 20 microseconds, the
recording of an image occurs in 256.times.20 microseconds, i.e. in
about 5 milliseconds.
[0059] In the next scan (phase-delayed, next 256 lines), the
resolution for the immobile object will be doubled, while rapidly
moving objects appear out of focus. Scan undercuts are carried out
until the limit of the optical resolution is reached. According to
the Nysquist criteria, this limit is reached, when the sampling
increments correspond to half of the optical resolution of the
microscope. If for example to reach the Nysquist criteria 2048
lines must be scanned, the demand interval at which the structures
with high spatial resolution may be examined, is 2048.times.20
microseconds, i.e. 40 milliseconds.
[0060] Rapidly moving objects initially appear out of focus (due to
the lower spatial sampling resolution), once they remain in one
place during the process they appear more clearly due to the
repeated and offset scanning.
[0061] By recording with lower resolution and at a higher speed (as
an overview scan), rapid movements become visible, which would not
be visible in recordings with the highest resolution (due to the
duration of the image recording).
[0062] With the appropriate input instrument, for example a slider
(FIG. 6), the relationship between the temporal resolution At
(frame rate) of the recordable velocity V (as expected by the
user), with which the objects move through the scan field, and the
special resolution Ar, is adjusted. This always represents a
compromise, which may be optimized by the user according to his
expectations.
[0063] Rapidly moving objects measuring 100 micrometers may be
present; in this case, a resolution of 1 micrometer is not
necessary, the user could enter a resolution of 10 micrometers and
use the increase of the recording speed to improve the acquisition
of the object movements.
[0064] Objects which are static in comparison to the image rate of
the microscope are represented with optical resolution at the
diffraction limit.
[0065] Dynamic objects moving faster than the image rate are
represented with a spatial resolution of the sampling rate, which
is generally lower than the optical resolution.
[0066] When recording a time series, dynamic objects initially
appear out of focus, but as soon as they become static, they are
represented with the resolution at the diffraction limit.
[0067] Rapid dynamic objects may become visible by correlation of
individual images to each other. This is done by coloring
correlated points (of the images recorded successively) with one
color and the remaining points with a different color.
[0068] A color coded overlay of fast and slow moving objects may
occur, whereas the static image information may be separated by a
correlation of the images. The image points correlating at
different times are used for this purpose.
[0069] In FIG. 7a, a monitor image with high optical resolution is
represented.
[0070] On the right, in an enlarged section of the left figure
part, rapidly moving objects are represented, which are only
visible in one location due to the high optical resolution.
[0071] In FIG. 7b, the optical resolution has been lowered
according to the invention (lower image rate).
[0072] Hereby, faster moving objects (represented in exaggeration)
appear out of focus, whereas static objects continue to appear
clearly. The movements of the unclear objects may be observed and
recorded.
[0073] The creation of images with a reduced image rate is
explained in FIG. 8.
[0074] Line detector is located on x-axis, shifting on y-axis,
signals used for the formula (Ck,i) j shift increment is vertical
(in y-direction).
[0075] The measured signals in individual channels are marked with
(ckij)j, whereas i=1 . . . N is the channel number of the line
detector, k is the number of lines and j=0 . . . n-1 is a multiple
of the shift a/n. Per column, for the calculation of the N times n
values Sm, differences of sums are calculated for individual values
according to the following algorithm:
S 1 = c 1 , 0 ' = i = 1 N c i , 0 - i = 1 N - 1 c i , 1
##EQU00001## S 2 = c 1 , 1 ' = i = 1 N c i , 1 - i = 1 N - 1 c i ,
2 ##EQU00001.2## ##EQU00001.3## S n - 1 = c 1 , n - 2 ' = i = 1 N c
i , n - 2 - i = 1 n - 1 c i , n - 1 ##EQU00001.4## S n = c 1 , n -
1 ' = i = 1 N - 1 c i , n - 1 - i = 2 N c i , 0 - m = 1 n - 2 c N ,
m ##EQU00001.5## ##EQU00001.6## S k n + 1 = c k , 0 ' = i = k N c i
, 0 - i = k N - 1 c i , 1 ##EQU00001.7## S k n + 2 = c k , 1 ' = i
= k N c i , 1 - i = k N - 1 c i , 2 ##EQU00001.8## ##EQU00001.9## S
k n + j + 1 = c k , j ' = i = k N c i , j - i = k N - 1 c i , j + 1
##EQU00001.10## ##EQU00001.11## S ( k + 1 ) n - 1 = c k , n - 2 ' =
i = k N c i , n - 2 - i = k N - 1 c i , n - 1 ##EQU00001.12## S ( k
+ 1 ) n = c k , n - 1 ' = i = k N - 1 c i , n - 1 - i = k + 1 N - 1
c i , 0 - m = 1 n - 2 c N , m ##EQU00001.13## ##EQU00001.14##
S.sub.Nn-n=c.sub.N,0'=c.sub.N,0
S.sub.N-n-n-1=c.sub.N,1'=c.sub.N,1
. . .
S.sub.Nn=c.sub.N,n-1'=c.sub.N,n-1
[0076] The calculated S values (interim values per column) may then
be graphically represented on the indicated image, e.g. by means of
a scan.
[0077] FIG. 9 shows the connection between the detector resolution
and the number of shifts n based on the configuration described
above. For n=1, the spatial resolution of the detection unit equals
the spatial resolution of the increment (a). For 5 shifts at a/s,
the spatial resolution of the detection unit is a/5. The maximum
spatial resolution which may be achieved is determined by an
optical limit resolution of the microscope. According to the scan
theorem by Nyquist, this maximum spatial resolution
(.quadrature..quadrature.) is reached exactly when the detector
resolution is equal to half of the potential resolution of the
microscope (.quadrature..quadrature.). This corresponds to a
number:
n max = 2 L .DELTA. .rho. ##EQU00002##
[0078] In the strip projection (7505), partial images are recorded
and calculated and thus a higher resolution is achieved. If these
partial images were utilized to obtain information, they could be
used with a lower local resolution but a higher temporal resolution
(e.g. three times faster).
[0079] Image information from the recorded partial images could
thus be obtained, whereas by interpolation a scan of the image
could be equalized, which could at the same time provide
information on the rapid movements in the image. The grid could
here be hidden by averaging over a grid period or by evaluating the
maximum points.
[0080] FIG. 2 shows a further definition of the invention.
[0081] If, due to the increased acquisition velocity of specimens
with a confocal microscope, lines or images are skipped,
fluorescence specimens show an irregular bleaching or strong
bleaching of certain regions. With the procedure described here, a
uniform bleaching of the specimen may be achieved.
[0082] To increase the recording velocity in the acquisition of
images with confocal microscopes, only every n-th line is
illuminated and recorded (FIG. 10). The intensity values for the
skipped lines are interpolated from the intensities of neighboring
pixels. In time series and continuous data acquisition, individual
lines in a fluorescence specimen are bleached, while the
neighboring regions are not bleached.
[0083] A reference acquisition here describes an image acquisition,
where the metric distance of the recorded pixels is equal in both
image directions. The image directions are designated with x- and y
direction, whereas the x-direction in punctual illumination of the
specimen is the direction in which the point is rapidly moved
during the scan of the specimen. In an illumination of the specimen
line by line, the x-direction shall be the direction of the line.
The y-direction shall be positioned vertically to the x-direction
and in the image plane.
[0084] A whole-numbered nesting value n is determined, which
indicates how many lines are skipped during the accelerated data
acquisition in y-direction. During a repeated acquisition of an
image, the specimen will not be scanned in the same spot but rather
shifted by a certain amount in y-direction. The amount of shifting
may be different for every line. However, the procedure is simpler,
if the same amount is used. In the simplest case, the amount of
shifting corresponds to the value of the line distance in
y-direction from the reference image, and illuminated in the n*i-th
image acquisition with whole-numbered i in the same spot as in the
first image.
[0085] With this procedure, a uniform bleaching of the specimen is
achieved. The maximum bleach effect for individual cells may be
reduced approximately by the factor 1/n.
[0086] The procedure may be used for the acquisition of images with
image planes of any orientation relative to the illumination
direction. Apart from scanners and piezo drives, other drives may
be used for the shifting.
[0087] For the acquisition of image batches, a procedure may be
used which is based on the same idea and simply represents a
generalization on a further dimension.
[0088] During this process, individual images of the batch recorded
during the next acquisition of the batch are shifted vertically to
the image plane. The procedures of the nested acquisition of images
and batches may also be used simultaneously.
[0089] The invention is not based on the line by line scanning. In
Nipkow scanners, the evaluation of a part of the perforated spirals
or other perforated configurations could be eliminated in an
initial step and then other perforated configurations could be used
in a further step.
[0090] In multipoint configurations, that are moved over a
specimen, certain point areas or point lines could be used for
evaluation.
[0091] The described invention represents a considerable increase
of possibilities of use of rapid confocal laser scan microscopes.
The significance of such a further development may be analyzed
according to the standard literature on cell biology and the rapid
cellular and sub-cellular procedures1 described therein as well as
the used research methods with a number of dyes2.
[0092] See:
[0093] .sup.1B. Alberts et al. (2002): Molecular Biology of the
Cell; Garland Science.
[0094] .sup.1,2G. Karp (2002): Cell and Molecular Biology: Concepts
and Experiments; Wiley Text Books.
[0095] .sup.1,2R. Yuste et al. (2000): Imaging neurons--a
laboratory Manual; Cold Spring Harbor Laboratory Press, New
York.
[0096] .sup.2R. P. Haugland (2003): Handbook of fluorescent
Specimens and research Products, 110th Edition; Molecular Specimens
Inc. and Molecular Specimens Europe BV.
[0097] The invention is particularly important for the following
processes and procedures:
[0098] Development of Organisms
[0099] The described invention may be used, among other things, for
the analysis of development processes, which are mainly
characterized by dynamic processes from one tenth of a second up to
an hourly range. Examples used on the level of united cell
structures and whole organisms are described herein among other
things:
[0100] In 2003, Abdul-Karim, M. A. et al. described in Microvasc.
Res., 66:113-125 along term analysis of blood vessel changes in
live animals, whereas fluorescence images were recorded in
intervals over several days. The 3D data acquisitions were
evaluated with adaptive algorithms, in order to schematically
represent movement trajectories.
[0101] In 2003, Soll, D. R. et al. described in Scientific World
Journ. 3:827-841a software based movement analysis of microscopic
data of nuclei and pseudopodia of live cells in all 3 spatial
dimensions.
[0102] In 2002, Grossmann, R. et al. described in Glia, 37:229-240
a 3D analysis of the movements of microglia cells in rats, whereas
the data was recorded over a period of up to 10 hours. At the same
time, after traumatic damage, fast reaction of the Glia could be
observed, so that a high data rate and corresponding data volume
results.
[0103] This particularly concerns the following key points:
[0104] Analysis of live cells in a 3D environment, the neighboring
cells of which react sensitively to laser and must be protected
from the illumination in the 3D-ROI;
[0105] Analysis of live cells in a 3D environment with markings
which shall be bleached systematically by laser in 3D, e.g., z.B.
FRET experiments;
[0106] Analysis of live cells in a 3D environment with markings
which are bleached systematically by laser and shall be observed at
the same time outside of the ROI, e.g. FRAP- and FLIP-experiments
in 3D;
[0107] Systematic analysis of live cells in a 3D environment with
markings and drugs showing manipulation determined changes by laser
illumination, e.g. activation of transmitters in 3D;
[0108] Systematic analysis of live cells in a 3D environment with
markings showing manipulation determined color changes by laser
illumination, e.g. paGFP, Kaede;
[0109] Systematic analysis of live cells in a 3D environment with
very weak markings, which require an optimal balance of confocality
against detection sensitivity.
[0110] Live cells in a 3D tissue compound with varying multiple
markings, e.g. CFP, GFP, YFP, DsRed, HcRed or similar.
[0111] Live cells in a 3D tissue compound with markings showing
function determined color changes, e.g. Ca+ markers
[0112] Live cells in a 3D tissue compound with markings showing
development determined color changes, e.g. transgenic animals with
GFP
[0113] Systematic analysis of live cells in a 3D environment with
markings showing manipulation determined color changes by laser
illumination, e.g. paGFP, Kaede
[0114] Systematic analysis of live cells in a 3D tissue compound
with very weak markings, which require a restriction of confocality
in favor of the detection sensitivity.
[0115] The last named point is combined with the preceding.
[0116] Transportation Processes in Cells
[0117] The described invention is excellent for the examination of
intracellular transportation processes, since relatively small
motile structures, e.g. proteins, with high speed, must be
represented here (mostly in the area of hundredth of seconds). In
order to record the dynamics of complex transportation processes,
applications such as FRAP with ROI bleaches are often used.
Examples for such studies are described in the following:
[0118] In 2000, Umenishi, F. et al. described in Biophys J.,
78:1024-1035 an analysis of the spatial mobility of Aquaporin in
GFP transfected culture cells. For this purpose, points in the cell
membrane were systematically and locally bleached and the diffusion
of the fluorescence was analyzed in the environment.
[0119] In 2002, Gimpl, G. et al. described in Prog. Brain Res.,
139:43-55 experiments with ROI bleaches and fluorescence imaging to
analyze the mobility and distribution of GFP marked Oxytocin
receptors in fibroblasts. This poses high requirements to the
spatial positioning and resolution as well as the direct temporal
consequence of bleaching and imaging.
[0120] In 2001, Zhang et al. described in Neuron, 31:261-275 live
cell imaging of GFP transfected nerve cells, whereas the movement
of granuli was analyzed by combining bleach and fluorescence
imaging. The dynamics of the nerve cells places high requirements
to the velocity of the imaging.
[0121] Interaction of Molecules
[0122] The described invention is particularly convenient to
represent molecular and other sub-cellular interactions. Herein,
very small structures with high velocity (in the area of hundredth
of seconds) must be represented. In order to dissolve the spatial
position of the molecules required for the interaction, indirect
techniques such as FRET with ROI bleaches are used.
[0123] Used examples are described in the following:
[0124] In 2004, Petersen, M. A. and Dailey, M. E. describe in Glia,
46:195-206 a dual channel acquisition of live hippocampus cultures
in rats, whereas two channels were recorded spatially in 3D and
over a longer period of time for the markers Lectin and Sytox.
[0125] In 2003, Yamamoto, N. et al. described in Clin. Exp.
Metastasis, 20:633-638 a two color imaging of human fibrosarcoma
cells, wherein green and red fluorescent protein (GFP and RFP) were
observed simultaneously in real time.
[0126] In 2003, Bertera, S. et al. described in Biotechniques,
35:718-722 a multicolor imaging of transgenic mice marked with
timer reporter protein, which changes its color from green to red.
The image acquisition occurs as fast series 3-dimensionally inside
the tissue of a live animal.
[0127] Signal Transfer Between Cells
[0128] The described invention is excellent for the examination of
extremely fast signal transfer procedures. These often
neurophysiological processes pose high requirements to the temporal
resolution, since the activities transmitted by ions are in the
range of hundredths to less than thousandths of seconds. Used
examples of studies of the muscle and nerve system are described
here:
[0129] In 2000, Brum G et al. described in J Physiol. 528: 419-433
the localization of rapid Ca+ activities in the muscle cells of a
frog after stimulation with caffeine as a transmitter. The
localization and micrometer precise resolution was only possible
due to the use of a fast confocal microscope.
[0130] In 2003, Schmidt H et al. described in J Physiol. 551:13-32
an analysis of Ca+ ions in nerve cell appendages of transgenic
mice. The study of rapid Ca+ transients in mice with altered Ca+
binding proteins could only be carried out with the help of high
resolution confocal microscopy, since a localization of the Ca+
activity within the nerve cell and its exact temporal kinetics play
an important role.
* * * * *