1. Introduction. Received 13 August 1998: accepted 4 November Correspondence to: C. Cremer Fax:

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1 International Journal for Light and electron Optics Confocal laser scanning fluorescence microscopy: In situ determination of the confocal point spread function and the chromatic shifts in intact cell nuclei P. Edelmann 1,2 A. Esa 1, M. Hausmann 1, C. Cremer 1,2 1 Applied Optics and Information Processing, Institute of Applied Physics, University of Heidelberg, Germany 2 Interdisciplinary Centre of Scientific Computing, University of Heidelberg, Germany Abstract: The confocal point spread function (PSF) was measured in three dimensionally (3D ) conserved nuclei of human cells using point like fluorescence labels coupled specifically to small chromatin regions. Since the resolution of a confocal fluorescence microscope, that is the smallest routinely measurable distance using one spectral signature (fluorochrome) only, is normally given by one full width at half maximum (FWHM) of the PSF. the results indicate that distance measurements in cell nuclei under the suboptimal optical conditions relevant here are usually limited to a regime of about >0.30 µm in lateral and > µm in axial directions. Using two or more spectral signatures, however, to label point like objects, the technique of Spectral Precision Distance Microscopy (SPDM) allows the determination of distances below the above mentioned resolution criterion. SPDM requires the exact determination of chromatic shifts under optical conditions close to the actual condition used in the precision distance measurements. Here, high precision chromatic shift measurements in situ in cell nuclei are presented, opening the avenue to distance measurements far below the normal resolution limit. Thus. for objects located :n the interior of intact cell nuclei, a "resolution equivalent" of some tens of nanometers can be realised. 1. Introduction The imaging properties of a confocal laser scanning fluorescence microscope can be described by its (three dimensional) point spread function (PSF) [15]. Under the assumption that imaging is linear and shift invariant [18]. The knowledge of the PSF is sufficient to obtain the image which should result from any known object. Furthermore, the PSF can be used to improve the estimate of an imaged object by deconvolution of the image 14). In a perfect optical system the PSF can be calculated assuming ideal optical conditions. Under practical conditions, however, several factors such as the refractive index mismatch of the components of the optical pathway, the refraction index variation within the specimen, the fluorescence photon statistics, the voxelisation and the digitisation of the image may lead to deviations from the ideal conditions and reduction of both contrast and Optik 110, No.4(1999) Urban & Fischer Verlag Received 13 August 1998: accepted 4 November Correspondence to: C. Cremer Fax: resolution. Thus. a calculated PSF often is not very useful to reconstruct object contours from a real image. In this case it is helpful to measure the PSF in the respective specimen in situ, so that optical insufficiencies deriving from real objects can be overcome [ 14. In fluorescence confocal laser scanning microscopy, biological specimens are typically objects under such suboptimal conditions. For instance, cell nuclei with chromatin regions specifically labelled with fluorochromes by the FISHtechnique (FISH = fluorescence in situ hybridisation) [10] are examples for quantitative 3D microscopy. Especially, to investigate the functional organisation of the genome (e.g., [4]), precise measurements of distances and angles between small chromatin regions situated in the interior of SD conserved nuclei are required. Here, we present in situ measurements of the confocal point spread function in such 3D conserved cell nuclei in order to express the "real" resolution limits determined by the full width at half maximum (FWHM) of the PSF. A short genomic sequence of 40kb (kb = kilo base pairs of the DNA polynucleotide chain) was labelled with one fluorochrome by FISH in interphase nuclei of bone marrow cells. Due to the complex folding of the chromatin in the interphase nucleus, this sequence formed a small sub resolution, i.e. point like, object. Since the standard expression for resolution is related to the FWHM of the PSF, the discrimination of point like objects (e.g., two fluorochrome labelled genomic target sequences in the interphase nucleus) and distance measurements between them are restricted to distances 1 FWHM if both objects carry the same spectral signature (= fluorochrome labels of the same relevant spectral characteristics). The determination of distances far below the resolution limit is possible by applying the concept of Spectral Precision Distance Microscopy (SPDM) [1] [3] [7] [5]. In contrast to the surface based methods of Scanning Near Field Optical Microscopy, SPDM allows to obtain information on the nanostructure of

2 195 fluorescent objects located in the interior of thick transparent specimen without micromechanical sectioning: Using a) point like objects labelled with different spectral signatures and b), spectrally differential registration of images, the intensity bary centres can precisely be localised independently for each of these objects in the diffracted /99/110/ $12.00/0 images. After a careful correction of optical aberrations especially chromatic aberrations (chromatic shifts) which exist also for high quality microscope lenses (e.g an apochromatic lens is completely corrected only for three wavelengths values), the determination of distances much smaller than the optical resolution (as given by the FWHM) becomes feasible. The "resolution equivalent (RE)" for distance measurements defined by the smallest measurable distance between pointlike objects of different spectral signatures. is mainly controlled by the localisation accuracy (accuracy to determine the intensity bary centres), and the accuracy of monochromatic and especially of chromatic aberration calibration. Recently, it has been shown that the calibration of monochromatic aberrations in both lateral and axial direction can be done very precisely using quartz glass beads with fluorescent cores 11 ] or with the help of micro axial tomography [13]. In addition, it has been shown that a localisation accuracy of about 20 nm for test particles and about nm for labelling sites in cell nuclei can be obtained in confocal laser scanning microscopy[2]. Since the chromatic aberrations are dependent on the optical micro conditions, optimum determination of chromatic shift has to be done in situ. Here, we used again a short genomic sequence of a 115 kb DNA fragment simultaneously labelled with three fluorochromes (SpectrumGreen, SpectrumOrange and SpectrumRed), equivalent to fluoresceine (FITC), rhodamine (TRITC) and cyanine (CY5) in human bone marrow interphase nuclei. 2. Materials and Methods 2.1. Preparation of nuclei with point like fluorescent objects For the determination of the fluorescence wavelength dependent PSF a cosmid DNA sequence (cos 8 abl) of 40 kb which maps to the q34.1 band of chromosome 9 was labelled along its full length either with fluoresceine isothiocyanate (FITC) 12 dutp, or with biotin 14 dctpor with digoxigenin 11 dutp by a Random Priming Reaction according to the manufacture's instruction (Life Technology, Cibco BRL). Fluorescence detection was performed either directly (FITC) or after immunohistochemistry with cyanine 5 (CY5) conjugated avidin against biotin (Amersham Life Science), or tetramethylrhodamine isothiocyanate (TRITC) conjugated antidigoxigenin (Boehringer Mannheim). For the determination of the chromatic shift a YAC DNA sequence (B99E11) of 115 kb was simultaneously labelled with three different fluorochromes with different spectral signatures by a nick translation kit according to the manufacture's instruction (Vysis Germany). For the determination of the chromatic shift it is highly desirable to incorporate the different fluorochromes in comparable amounts on the same DNA sequences. For this purpose, three different fluorochromes, SpectrumGreen. SpectrumOrange and SpectrumRed (equivalent to FITC, TRITC, CY5) (1:1:1;v:v:v) were used. Fluorescence in situ hybridisation (FISH) using the fluorescence labelled probes described above was applied to bone marrow interphase nuclei of a leukemia patient (cells kindly provided by Dr. L Trakhtenbrot. Institute of Hematology. Tel Hashomer, Israel; probes kindly provided by Dr. U. Weier. University of Berkeley, California). Preparations of interphase nuclei and FISH were performed according to a standard protocol [6]. To reduce photo bleaching, slides were mounted in antifade containing the fluorochrome 4,6 diamidino 2 phenylindole (DAPI) (1 Hg/ml). In figure 1 the described procedure is schematically depicted for the case of simultaneous three color labelling 2.2. Image acquisition and image processing For image acquisition the cell nuclei were directly mounted under the coverglas with Vecta Shield antifade solution. Series of light optical sections of the cell nuclei were recorded with a three channel Leica TCSJMT confocal laser scanning microscope (Leica Lasertechnik, Heidelberg) equipped with a Plan Apo 63x/1.4 oil objective. The fluorochromes were excited by the three lines of an argon kryptonlaser switched by an AOTF (λ iö = 488 nm for FITC/ Spectrum Green; λ iö = 568 nm for TRITC/SpectrumOrange; λ iö = 647 nm for CY5/SpectrumRed). The light emitted by the fluorochromes was recorded separately by independent photomultiplier channels. a) PSF measurements For the in situ determination of the PSF, stacks of equidistant (0.08 µm) 8 bit greyscale images were obtained with a voxel size of 0.05 µm in x and y

3 196 Ip 665 nm for CY5). Considering the maxima of the emission spectra of the fluorochromes used ( FITC 520 nm; TRITC 578 nm; CY5 672 nm) the wavelength of highest Fig. 1. Simultaneous labelling of the same small genomic target sequence with three different fluorochromes (triplelabelling) using a standard Nick translation kit. Fig. 2. a) In situ mean lateral confocal point spread function of the TRITC channel in human cell nuclei. The raw data are given by points, the closed line shows the result of a fit the theoretical curve used for the determination of the full width at half maximum; b) In situ mean axial confocal point spread function of the TRITC channel in human cell nuclei. The raw data are given by points, the closed line shows the result of a polynomial fit used for the determination of the full width at half maximum. The dashed line shows the result of a fit with the theoretical aberration free and therefore symmetrical curve (according to [9]). detectable fluorescence intensity was fixed at λ det = 520nm for FITC; λ det = 590 nm for TRITC and λ det = 672 nm for CY5. For the evaluation of the registered images, the labelled sub resolution sites were extracted by interactively setting of a sub volume containing 40x40x40 voxels. Background noise in the data mainly introduced by scattering and autofluorescence was reduced by the following semi automated procedure. In every processed nucleus an additional sub volume was extracted which contained only background and no spots. In each consecutive image plane, the mean grey value of these background data was determined and subtracted from each voxel of the corresponding plane in the sub volume containing the extracted spot from the same nucleus. For each fluorochrome, spots were recorded and their images were averaged by overlaying all the brightest voxels of the extracted spot stacks. Overlying means that for each spot, the positions of the maxima of the individual PSF curves were assumed to coincide with the positions of the brightest voxel. Since all images of the same fluorochrome labelled regions were recorded using the same laser power, filtersettings, pinhole size (0.75x airy disk produced in the pine hole plane by a point like (FITC) labelled object) and photo multiplier voltage, every grey value represented the same number of detected photons. Therefore no normalisation of intensities was necessary prior to averaging of spots. b) Chromatic shift measurements For the in situ determination of chromatic shifts, stacks of equidistant (0.21 µm) 8 bit grayscale images each were simultaneously recorded for the three fluorochromes used. The voxel size was 0.08 µm in x and y direction. The fluorochromes were excited with the appropriate laser lines and detected simultaneously by the following filter settings: SpectrumGreen (emission maximum 524 nm) by a 530/30 nm bandpass filter; SpectrumOrange (emission maximum 558 nm) by a 600/30 nm bandpass filter and SpectrumRed (emission maximum 612 nm) by

4 197 a 665 nm longpass filter. The triple labelled sites were segmented by interactively setting of an individual threshold for each fluorescence detection channel. Global background subtraction was performed by subtracting the mean grey value of the whole data stack. All connected voxels of the spots were identified using the 26 connectivity rule and then used to calculate the position of the bary centre of intensity of each spot for the three channels. Since the labelling molecules were randomly distributed along the DNA sequence hybridized, their intensity bary centers should ideally coincide. Thus, chromatic shifts were calculated as the distances of the positions of the intensity bary centres of the individual spots in the three colour channels. 44 spots located within the cell nuclei were recorded and the chromatic shifts were averaged. 3. Results 3.1. Confocal in situ PSF The confocal PSF was determined by FISH labelling of a genomic target sequence of 40 kb with a subresolution size in interphase nuclei. For each fluorochrome, about 15 20

5 198 SpectrumGreen, SpectrumOrange and sub resolution spots were averaged after overlaying of the brightest voxel of each spot. Therefore, for each fluorochrome an averaged confocal PSF was determined. Lateral line scans of these PSFs could well be fitted witli the theoretical aberration free curve [9 by means of the Levenberg Marquart Algorilhm (see Fig. 2a): where r is the radial coordinate in the lateral plane, Io the maximum intensity and b a constant, J 1, the first order Bessel function, λ ill the illumination wavelength and λ det the detection wavelength. The parameters Io and b were adjusted by the fitting algorithm. This equation was used to determine the lateral FWHM of each averaged in situ 3D PSF: FITC : 283 nm TRITC : 278 nm CY5 : 326 nm Line scans of the PSFs along the optical axis (fig. 2b) revealed that the axial response was slightly asymmetric and did not well fit to the axial theoretical curves given by [9]. This agrees with theoretical investigations on the effect of spherical aberrations which predicted an increasing asymmetric axial response with increasing refractive index mismatch [17] and increasing depth of focus [8], [i5j. Therefore. we used a polynomial fit to obtain a smooth curve and to determine the axial FWHM of each averaged in situ 3D PSF: 3.2. In situ chromatic shift FITC :718nm TRITC: 764 nm CY5 :613nm The chromatic shift was analysed by using triplelabelled sites. Here, the three fluorochromes SpectrumRed were used for simultaneous labelling of a nuclear DNA sequence of 115kb. A total number of 44 spots located within human cell nuclei were recorded. From the labelling technique, under ideal optical conditions tree from chromatic aberration, the intensity bury centres of all three colours should colocalise. Under the real optical conditions used here. however, experimentally small distances between these bary centers were measured. Thus, the detected miscolocalisation revealed the chromatic aberration shift. Table 1 shows the experimental chromatic aberration shifts in situ. The lateral plane is represented by the x and y coordinates. Lateral shifts between about 10 nm and about 35 nm were obtained. The ; axis denotes the direction parallel to the optical axis. Relevant axial shifts of about ± 190 were measured, whereas a negative shift between the first fluorochrome and the second fluorochrome means that the first fluorochrome is closer to the coverglass than the other one. 4. Discussion The determination of the confocal PSF in situ by using a short genomic DNA sequence. which for a given PSF measurement was FISH labelled with one fluorochrome revealed, that the shape of the lateral PSF was not apparently affected by the specimen induced aberrations or by aberrations caused by refractive index mismatch under the conditions used. However, the FWHM did not clearly show the expected wavelength dependent behaviour. This is probably caused by the fact that in the TRITC channel the highest number of photons was detected (our own calibration measurements, data not shown). Furthermore, the LEICA TCSNT confocal laser scanning microscope uses one pinhole only to image all colour channels. Size and position of the pinhole, however, can only be ideal for one channel, particularly in the presence of chromatic aberrations. The axial response was influenced by aberrations, resulting in an asymmetric response.

6 199 However, the loss in resolution and symmetry was a minor one, since the biological objects (cell nuclei) were mounted directly under the coverglass. Again, the wavelength dependent behaviour of the FWHM did nor clearly follow theoretical expectations. The chromatic shift in situ analysis presented in this paper is a further experimental improvement in comparison to recently presented methods [11], [12], [1] using multi spectral fluorescence beads. In this latter case the most obvious setback is that using such beads mounted under the cover glass does nut consider the imaging conditions inside real biological objects, in this case cell nuclei. Using FISH labelling spots as micro test objects instead of micro beads has revealed practical advantages, because in order to obtain the real optical microconditions inside cell nuclei, micro injection of multi spectral beads into cell nuclei is required. However. under routine conditions this appears unpractical for laboratory purposes because of the exhausting effort, time and cost needed to prepare and to record the necessarily large number of cells. Moreover, the numbers of microbeads injected simultaneously into a cell nucleus cannot be controlled easily so that the size of the sub resolution object (individual bead or beadcluster) and consequently its intensity cannot be determined. Furthermore, typical commercially available multi spectral latex beads neither have the same excitation spectra nor the same emission spectra as the labelling fluorochromes used for biologically relevant FISH investigations. Different excitation and emission spectra, however, should result in different chromatic shifts. The concept of spectral precision distance microscopy (SPDM) in the study of human genome structure is based on the requirement that for an optimum resolution equivalent the chromatic shifts are calibrated precisely under the same micro optical conditions in which the biological measurements take place. This makes it highly desirable to use the same fluorescence absorption and emission spectra in the same object, i.e. in situ, for calibration as well as for biological structure analysis. In general, the same principles can be applied to all other microscopic studies of the distribution of point like targets in transparent specimens using multicolour fluorescence labelling. Acknowledgement. The authors thank Dr. L. Trakhtenbrot, Institute of Hematology, The Chaim Sheba Medical Center, Tel Aviv University, Tel Hashomer, Israel, for providing the cells. Spectral Precision Distance Microscopy (SPDM) is a patent application by C. Cremer, M. Hausmann. J. Bradl and B. Rinke. The financial support of the German Federal Minister of Science, Education, Research and Technology (BMBF) is gratefully acknowledged. A. Esa receives a scholarship of the Friedrich Ebert Stiftung. References [1] Bomfleth H, Satzler K, Eils R. Cremer C: High precision distance measurements and volumeconserving segmentation of objects near and below the resolution limit in three dimensional confocal fluorescence microscopy. J Microsc 189 (1998) [2] Bradl J, Rinke B, Esa A, Edelmann P. Krieger H, Schneider B, Hausmann M, Cremer C: Comparative study of three dimensional localization accuracy in conventional, confocal laser scanning and axial tomographic fluorescence light microscopy. Proc. SPIE. (1996 ) 2926, pp [3] Cremer C, Hausmann M, Bradl J. Rinke B: Verfahren zurmultispektralen Präzisionsdistanzmessung in biologischen Mikroobjekten. German patent application. (1996) No [4] Cremer C. Münkel V. Granzow M. Jauch A. Dietzel S, Eils R. Guan X Y. Meltzer PS. Trent JM. Langowski J. 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