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Here we describe an automated method, named serial two-photon (STP) tomography, that achieves high-throughput fluorescence imaging of mouse brains by integrating two-photon microscopy and tissue sectioning. STP tomography generates high-resolution datasets that are free of distortions and can be readily warped in three dimensions, for example, for comparing multiple anatomical tracings. This method opens the door to routine systematic studies of neuroanatomy in mouse models of human brain disorders.

Image cytometry technology has been extended to 3D based on high-speed multiphoton microscopy. This technique allows in situ study of tissue specimens preserving important cell-cell and cell-extracellular matrix interactions. The imaging system was based on high-speed multiphoton microscopy (HSMPM) for 3D deep tissue imaging with minimal photodamage. Using appropriate fluorescent labels and a specimen translation stage, we could quantify cellular and biochemical states of tissues in a high throughput manner. This approach could assay tissue structures with subcellular resolution down to a few hundred micrometers deep. Its throughput could be quantified by the rate of volume imaging: 1.45 mm(3)/h with high resolution. For a tissue containing tightly packed, stratified cellular layers, this rate corresponded to sampling about 200 cells/s. We characterized the performance of 3D tissue cytometer by quantifying rare cell populations in 2D and 3D specimens in vitro. The measured population ratios, which were obtained by image analysis, agreed well with the expected ratios down to the ratio of 1/10(5). This technology was also applied to the detection of rare skin structures based on endogenous fluorophores. Sebaceous glands and a cell cluster at the base of a hair follicle were identified. Finally, the 3D tissue cytometer was applied to detect rare cells that had undergone homologous mitotic recombination in a novel transgenic mouse model, where recombination events could result in the expression of enhanced yellow fluorescent protein in the cells. 3D tissue cytometry based on HSMPM demonstrated its screening capability with high sensitivity and showed the possibility of studying cellular and biochemical states in tissues in situ. This technique will significantly expand the scope of cytometric studies to the biomedical problems where spatial and chemical relationships between cells and their tissue environments are important.

A two-pulse experiment is described, using stimulated emission to reduce the fluorescence of 1-ethyl-4-(4-(p-dimethylaminophenyl)-1,3-butadienyl)-pyridinium perchlorate (Pyridine 2) in a microscope of high numerical aperture. The experiment employed a 130 fs pulse at 375 nm for excitation and a 20 ± 5 ps pulse at 750 nm for stimulated emission. The pulses were provided by a mode-locked Ti:sapphire laser. The 375 nm excitation pulse was obtained by frequency-doubling and the 20 ± 5 ps infrared pulse by optical grating dispersion. The population of the excited state was monitored by varying the temporal delay between the excitation and stimulating pulse. This method enabled the measurement of the lifetime of the dye. For Pyridine 2 in glycerol, we determined a lifetime of 0.86 ± 0.2 ns. The decrease of fluorescence was due to stimulated emission, as was established by measurements of the temporal behaviour of the fluorescence signal upon amplitude modulation of the stimulating beam. We present a theoretical analysis of the temporal behaviour of depletion by stimulated emission in a two-pulse experiment.

We show experiments proving the feasibility of scanning fluorescence microscopy by three-photon excitation. Three-photon excitation fluorescence axial images are shown of polystyrene beads stained with the fluorophore 2,5-bis(4-biphenyl)oxazole (BBO). Three-photon excitation is performed at an excitation wavelength of 900 nm and with pulses of 130 fs duration provided by a mode-locked titanium sapphire laser. Fluorescence is collected between 350 and 450 nm. The fluorescence image signal features a third-order dependence on the excitation power, also providing intrinsic 3-D imaging. The resolution of a three-photon excitation microscope is increased over that of a comparable two-photon excitation microscope.

We introduce a method employing ferroelectric monomolecular layers, by which it is possible to measure the light field polarization in the focus of a lens. This method allowed us to noninvasively establish the perpendicularly oriented focal field that is anticipated at high apertures. For a numerical aperture 1.4 oil immersion lens illuminated with linearly polarized plane waves, the integral of the modulus square of the perpendicular component amounts to (1.51±0.2) % of that of the initial polarization. It is proven that depolarization decreases with decreasing aperture angle. Whereas for regular imaging conditions depolarization is largely negligible, it plays a significant role in microscopy of highest resolution, microspectroscopy, and single molecule studies.

Multifocal multiphoton microscopy (MMM) enhances imaging speed by parallelization. It is not well understood why the imaging depth of MMM is significantly shorter than conventional single-focus multiphoton microscopy (SMM). In this report, we show that the need for spatially resolved detectors in MMM results in a system that is more sensitive to the scattering of emission photons with reduced imaging depth. For imaging depths down to twice the scattering mean free path length of emission photons (2xl (s) (em)), the emission point spread function (PSF(em)) is found to consist of a narrow, diffraction limited distribution from ballistic emission photons and a broad, relatively low amplitude distribution from scattered photons. Since the scattered photon distribution is approximately 100 times wider than that of the unscattered photons at 2xl (s) (em), image contrast and depth are degraded without compromising resolution. To overcome the imaging depth limitation of MMM, we present a new design that replaces CCD cameras with multi-anode photomultiplier tubes (MAPMTs) allowing more efficient collection of scattered emission photons. We demonstrate that MAPMT-based MMM has imaging depth comparable to SMM with equivalent sensitivity by imaging tissue phantoms, ex vivo human skin specimens based on endogenous fluorophores, and green fluorescent protein (GFP) expressing neurons in mouse brain slices.

We studied the effect of electric field orientation on the point-spread function (PSF) of a 4Pi microscope. We show that in a standard 4Pi arrangement the orientation of the field can be used for changing between constructive- and destructive-mode 4Pi microscopy. The effect is counteracted by introduction of a phase shift of π into one of the half-arms. This compensation is compulsory during illumination with unpolarized or circularly polarized light. By performing our experiments with 1.2-N.A. water-immersion lenses, we demonstrate that water immersion is suitable for 4Pi confocal microscopy. At a two-photon excitation wavelength of 1064 nm, the water 4Pi confocal PSF features an axial lobe of 40% above and below the focal plane, which, by linear filtering, can be unambiguously removed. The measured axial full width at half-maximum of the PSF is 240 nm. This is 4.3 times narrower than its single-lens confocal counterpart. The 4Pi confocal microscope sets a new resolution benchmark in three-dimensional imaging of watery samples.

This review describes the development and some pilot applications of a new technology: 3D tissue image cytometry. 3D tissue image cytometry quantifies tissue morphology and biochemistry in a high throughput fashion. Promising applications range from cardiology, cancer biology, to tissue engineering. In this review, we will first examine traditional cytology, histology, and cytometry techniques that are the precursors of 3D tissue image cytometry. We will consider novel 3D optical imaging methods that form the technological basis for this new technology. Experiments for the validation and characterization of this instrument are presented. An application of this technology focusing on cardiac hypertrophy study is described in detail

Three-dimensional (3-D) tissue imaging offers substantial benefits to a wide range of biomedical investigations from cardiovascular biology, diabetes, Alzheimer's disease to cancer. Two-photon tissue cytometry is a novel technique based on high-speed multiphoton microscopy coupled with automated histological sectioning, which can quantify tissue morphology and physiology throughout entire organs with subcellular resolution. Furthermore, two-photon tissue cytometry offers all the benefits of fluorescence-based approaches including high specificity and sensitivity and appropriateness for molecular imaging of gene and protein expression. We use two-photon tissue cytometry to image an entire mouse heart at subcellular resolution to quantify the 3-D morphology of cardiac microvasculature and myocyte morphology spanning almost five orders of magnitude in length scales.

We developed 3D tissue image cytometry for the imaging of whole organs with subcellular resolution. This technique potentially allows tissue physiology to be understood on the basis of the underlying genomics and proteomics.

The development of high throughput tissue cytometry capable of mapping gene and protein expression profiles in 3-D with subcellular resolution throughout whole organs of small animals extends bioinformatic analysis to physiology and pathology.

High-speed two-photon imaging based on multi-foci excitation requires the use of spatially resolved detectors, such as charge coupled device (CCD) cameras, instead of single channel photomultiplier tube (PMT). The performance of systems based on both a PMT and a CCD in turbid medium was evaluated by measuring the image point spread function (PSF) and the image contrast as a function of depth and scattering coefficient with single point scanning. We found no significant change in the full-width at half maximum of the point spread function (PSF) for depth up to 100 μm. However, the CCD lost contrast significantly faster as a function of depth and increase scattering. This discrepancy is resolved by measuring a low amplitude but broad tail in the PSF distribution. The tail of the PSF distribution can be up to 200 μm in diameter. We further evaluate scattering effects in the imaging of GFP neurons in a mouse brain slice.

Publisher Summary This chapter describes a new cytometry technique based on the combination of Two-photon microscopy (TPM) and high-speed imaging techniques. TPM is a high-resolution fluorescence microscopy technique that provides 3D images of cells and tissues. It is well suited for imaging tissues. By incorporating high-speed image capabilities, it provides images of large populations of cells within their native tissue environment using TPM and extends standard 2D image cytometry into 3D. The native environment of cells is within the 3D environment of their host tissue but standard preparation for both flow and image cytometry involves the dissociation of cells from their tissue matrix. The chapter provides an overview of some of the early cytometry techniques such as flow, image, and tissue-based cytometry. Flow cytometry (FCM) is conducted with cells in suspension, whereas image cytometry studies are typically conducted in 2D culture. The biochemical, mechanical, and intracellular inputs present in a tissue affect the behavior of a cell. The underlying principles and instrumentation of two-photon tissue cytometry and some emerging applications of this technology are also described.

Summary form only given. We developed multi-photon fluorescence spectroscopy based on a 16/spl times/1 multianode photo multiplier tube and we continue to develop video rate microscopy based on an 8/spl times/8 multianode PMT. The spectroscopy consists of grating, 16/spl times/1 multianode PMT and a 16 channel photon counting card. It can take spectrum of pixels in addition to acquiring images. Multiple color fluorescence spheres, cells stained with multiple color fluorescence dyes and tissues were examined with the system. Video rate multi-photon microscopy is constructed based on an 8/spl times/8 micro lenslet array, an 8/spl times/8 multianode PMT and 64 photon counting card for deep tissue imaging which is difficult due to scattering of tissue samples.

By coherently adding the spherical wavefronts of two opposing lenses, two-photon excitation 4Pi-confocal fluorescence microscopy has achieved three-dimensional imaging with an axial resolution 3–7 times better than confocal microscopy. So far this improvement was possible only in glycerol-mounted, fixed cells. Here we report 4Pi-confocal microscopy of watery objects and its application to the imaging of live cells. Water immersion of 4Pi-confocal microscopy of membrane stained live Escherichia coli bacteria attains a 4.3-fold better axial resolution as compared to the best water immersion confocal microscope. The resolution enhancement results into a vastly improved threedimensional representation of the bacteria. The first images of live biological samples with an all-directional resolution in the 190–280 nm range are presented here, thus establishing a new resolution benchmark in live-cell microscopy. # 2001 Elsevier Science B.V. All rights reserved.

We propose and demonstrate a method employing ferroelectric monomolecular layers, by which it is possible to precisely measure the planar light field polarization in the focus of a lens. This method allowed us to establish for the first time to our knowledge, the perpendicularly oriented field that is anticipated at high apertures. For a numerical aperture 1.4 oil immersion lens illuminated with linearly polarized plane waves, the integral of the modulus square of the perpendicular component amounts to (1.51 r 0.2) % of that of the initial polarization. It is experimentally proven that depolarization decreases with decreasing aperture angle and increases when using annular apertures. Annuli formed by a central obstruction with a diameter of 89 % of that of the entrance pupil raise the integral to 5.5 %. This compares well with the value of 5.8% predicted by electromagnetic focusing theory; however, the depolarization is also due to imperfections connected with focusing by refraction. Besides fluorescence microscopy and single molecule spectroscopy, the measured intensity of the depolarized component in the focal plane is relevant to all forms of light spectroscopy combining strong focusing with polarization analysis.

By coherently adding the spherical wavefronts of two opposing lenses, two-photon excitation 4Pi-confocal fluorescence microscopy has achieved three-dimensional imaging with an axial resolution 3-7 times better than confocal microscopy. So far this improvement was possible only in glycerol-mounted, fixed cells. Here we report 4Pi-confocal microscopy of watery objects and its application to the imaging of live cells. Water immersion of 4Pi-confocal microscopy of membrane stained live Escherichia coli bacteria attains a 4.3-fold better axial resolution as compared to the best water immersion confocal microscope. The resolution enhancement results into a vastly improved three-dimensional representation of the bacteria. The first images of live biological samples with an all-directional resolution in the 190-280 nm range are presented here, thus establishing a new resolution benchmark in live-cell microscopy.

Electromagnetic focusing theory of light predicts that at high apertures field components arise that are polarized perpendicular to the initial polarization. Although vectorial depolarization has received considerable attention in focusing theory, no evidence has been presented as to its relevance in experiments. We measure the intensity of the perpendicularly orientated field in the focal region by utilizing monomolecular, fluorescent polydiacetylene layers whose transition dipoles are orientated in a single direction. For a 1.4 numerical aperture oil objective lens illuminated with linearly x-polarized light, we find that the integral of the modulus squared of the y-polarized focal field amounts to 1.5% of its x-polarized counterpart. In particular, we show here that the depolarization increases when using annular apertures. Annuli formed by a central obstruction with a diameter of 89% of that of the entrance pupil raise the integral to 5.5%. This compares well with the value of 5.8% predicted by electromagnetic focusing theory; however, the depolarization is also due to imperfections connected with focusing by refraction. Besides fluorescence microscopy and single molecule spectroscopy, the measured intensity of the depolarized component in the focal plane is relevant to all forms of light spectroscopy combining strong focusing with polarization analysis.

By combining the wavefronts produced by two high-aperture lenses, two-photon 4Pi-confocal microscopy allows three-dimensional imaging of transparent biological specimens with axial resolution in the 100–140-nm range. We reveal the imaging properties of a two-photon 4Pi-confocal microscope as applied to a fixed cell. We demonstrate that a fast, linear point deconvolution suffices to achieve axially superresolved 3D images in the cytoskeleton. Furthermore, we describe stringent algorithms for alignment and control of the two lenses. We also show how to compensate for the effects of a potential refractive index mismatch of the mounting medium with respect to the immersion system.

We introduce a multiphoton microscope for high-speed three-dimensional (3D) fluorescence imaging. The system combines parallel illumination by a multifocal multiphoton microscope (MMM) with parallel detection via a segmented high-sensitivity charge-couple device (CCD) camera. The instrument consists of a Ti-sapphire laser illuminating a microlens array that projects 36 foci onto the focal plane. The foci are scanned using a resonance scanner and imaged with a custom-made CCD camera. The MMM increases the imaging speed by parallelizing the illumination; the CCD camera can operate at a frame rate of 1428 Hz while maintaining a low read noise of 11 electrons per pixel by dividing its chip into 16 independent segments for parallelized readout. We image fluorescent specimens at a frame rate of 640 Hz. The calcium wave of fluo3 labeled cardiac myocytes is measured by imaging the spontaneous contraction of the cells in a 0.625 second sequence movie, consisting of 400 single images.

Summary We report the realization of stimulated emission on a microscopic scale. An experiment is presented describing significant depopulation of the excited state of the fluorophore Pyridine 2 with a mode-locked Ti: Sapphire laser. Stimulated emission is performed at 750 nm and excitation at the frequency doubled wavelength of 375 nm. The pulses are synchronized so that the stimulating pulse follows the excitation pulse after 5 ps. The set-up is a modified 4Pi-confocal microscope employing one of the objective lenses for excitation and the opposing one for stimulated emission.