Timothy Ragan

Serial two-photon tomography for automated ex vivo mouse brain imaging.

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.

Multifocal multiphoton microscopy based on multianode photomultiplier tubes

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.

High-resolution whole organ imaging using two-photon tissue cytometry

Three-dimensional (3-D) tissue imaging offers substantial benefits to a wide range of biomedical investigations from cardiovascular biology, diabetes, Alzheimers 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.

Three-dimensional tissue cytometer based on high-speed multiphoton microscopy.

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 mm3/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 1/105. 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.

In vivo and ex vivo tissue applications of two-photon microscopy.

Publisher Summary This chapter describes in vivo and ex vivo tissue applications of two-photon microscopy (TPM) and reviews key areas—such as neurobiology—in which TPM has already made a major impact. TPM is one of the emerging optical tissue imaging techniques, and it is constructive to compare two-photon imaging with other in vivo optical tissue imaging techniques. TPM is a powerful technique for tissue imaging because of its inherent 3D resolution and long penetration depth. This technique also provides biochemical information about tissues and causes minimal photodamage. TPM is based on a nonlinear fluorescence excitation process. The simultaneous absorption of two infrared photons promotes the transition of a fluorophore to the excited state. Because of the nonlinear excitation process, there is only sufficient photon density very near the focal region to produce appreciable excitation, which results in an inherent 3D sectioning effect. The unique advantages of TPM for deep tissue imaging have been recognized nearly since its inception. TPM tissue imaging applications are proliferating rapidly.

Two-Photon Tissue Cytometry

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.

High-Throughput Tissue Image Cytometry

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

Tissue image cytometry: whole organ imaging with subcellular resolution

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.