Excitation-scanning hyperspectral imaging microscope.

Hyperspectral imaging is a versatile tool that has recently been applied to a variety of biomedical applications, notably live-cell and whole-tissue signaling. Traditional hyperspectral imaging approaches filter the fluorescence emission over a broad wavelength range while exciting at a single band. However, these emission-scanning approaches have shown reduced sensitivity due to light attenuation from spectral filtering. Consequently, emission scanning has limited applicability for time-sensitive studies and photosensitive applications. In this work, we have developed an excitation-scanning hyperspectral imaging microscope that overcomes these limitations by providing high transmission with short acquisition times. This is achieved by filtering the fluorescence excitation rather than the emission. We tested the efficacy of the excitation-scanning microscope in a side-by-side comparison with emission scanning for detection of green fluorescent protein (GFP)-expressing endothelial cells in highly autofluorescent lung tissue. Excitation scanning provided higher signal-to-noise characteristics, as well as shorter acquisition times (300 ms/wavelength band with excitation scanning versus 3 s/wavelength band with emission scanning). Excitation scanning also provided higher delineation of nuclear and cell borders, and increased identification of GFP regions in highly autofluorescent tissue. These results demonstrate excitation scanning has utility in a wide range of time-dependent and photosensitive applications.

Thin-film tunable filters for hyperspectral fluorescence microscopy.

Hyperspectral imaging is a powerful tool that acquires data from many spectral bands, forming a contiguous spectrum. Hyperspectral imaging was originally developed for remote sensing applications; however, hyperspectral techniques have since been applied to biological fluorescence imaging applications, such as fluorescence microscopy and small animal fluorescence imaging. The spectral filtering method largely determines the sensitivity and specificity of any hyperspectral imaging system. There are several types of spectral filtering hardware available for microscopy systems, most commonly acousto-optic tunable filters (AOTFs) and liquid crystal tunable filters (LCTFs). These filtering technologies have advantages and disadvantages. Here, we present a novel tunable filter for hyperspectral imaging-the thin-film tunable filter (TFTF). The TFTF presents several advantages over AOTFs and LCTFs, most notably, a high percentage transmission and a high out-of-band optical density (OD). We present a comparison of a TFTF-based hyperspectral microscopy system and a commercially available AOTF-based system. We have characterized the light transmission, wavelength calibration, and OD of both systems, and have then evaluated the capability of each system for discriminating between green fluorescent protein and highly autofluorescent lung tissue. Our results suggest that TFTFs are an alternative approach for hyperspectral filtering that offers improved transmission and out-of-band blocking. These characteristics make TFTFs well suited for other biomedical imaging devices, such as ophthalmoscopes or endoscopes.

Tunable thin-film optical filters for hyperspectral microscopy.

Hyperspectral imaging was originally developed for use in remote sensing applications. More recently, it has been applied to biological imaging systems, such as fluorescence microscopes. The ability to distinguish molecules based on spectral differences has been especially advantageous for identifying fluorophores in highly autofluorescent tissues. A key component of hyperspectral imaging systems is wavelength filtering. Each filtering technology used for hyperspectral imaging has corresponding advantages and disadvantages. Recently, a new optical filtering technology has been developed that uses multi-layered thin-film optical filters that can be rotated, with respect to incident light, to control the center wavelength of the pass-band. Compared to the majority of tunable filter technologies, these filters have superior optical performance including greater than 90% transmission, steep spectral edges and high out-of-band blocking. Hence, tunable thin-film optical filters present optical characteristics that may make them well-suited for many biological spectral imaging applications. An array of tunable thin-film filters was implemented on an inverted fluorescence microscope (TE 2000, Nikon Instruments) to cover the full visible wavelength range. Images of a previously published model, GFP-expressing endothelial cells in the lung, were acquired using a charge-coupled device camera (Rolera EM-C2, Q-Imaging). This model sample presents fluorescently-labeled cells in a highly autofluorescent environment. Linear unmixing of hyperspectral images indicates that thin-film tunable filters provide equivalent spectral discrimination to our previous acousto-optic tunable filter-based approach, with increased signal-to-noise characteristics. Hence, tunable multi-layered thin film optical filters may provide greatly improved spectral filtering characteristics and therefore enable wider acceptance of hyperspectral widefield microscopy.

Optimization of flow cytometric detection and cell sorting of transgenic Plasmodium parasites using interchangeable optical filters.

BACKGROUND: Malaria remains a major cause of morbidity and mortality worldwide. Flow cytometry-based assays that take advantage of fluorescent protein (FP)-expressing malaria parasites have proven to be valuable tools for quantification and sorting of specific subpopulations of parasite-infected red blood cells. However, identification of rare subpopulations of parasites using green fluorescent protein (GFP) labelling is complicated by autofluorescence (AF) of red blood cells and low signal from transgenic parasites. It has been suggested that cell sorting yield could be improved by using filters that precisely match the emission spectrum of GFP. METHODS: Detection of transgenic Plasmodium falciparum parasites expressing either tdTomato or GFP was performed using a flow cytometer with interchangeable optical filters. Parasitaemia was evaluated using different optical filters and, after optimization of optics, the GFP-expressing parasites were sorted and analysed by microscopy after cytospin preparation and by imaging cytometry. RESULTS: A new approach to evaluate filter performance in flow cytometry using two-dimensional dot blot was developed. By selecting optical filters with narrow bandpass (BP) and maximum position of filter emission close to GFP maximum emission in the FL1 channel (510/20, 512/20 and 517/20; dichroics 502LP and 466LP), AF was markedly decreased and signal-background improve dramatically. Sorting of GFP-expressing parasite populations in infected red blood cells at 90 or 95% purity with these filters resulted in 50-150% increased yield when compared to the standard filter set-up. The purity of the sorted population was confirmed using imaging cytometry and microscopy of cytospin preparations of sorted red blood cells infected with transgenic malaria parasites. DISCUSSION: Filter optimization is particularly important for applications where the FP signal and percentage of positive events are relatively low, such as analysis of parasite-infected samples with in the intention of gene-expression profiling and analysis. The approach outlined here results in substantially improved yield of GFP-expressing parasites, and requires decreased sorting time in comparison to standard methods. It is anticipated that this protocol will be useful for a wide range of applications involving rare events.

3D single molecule tracking of quantum-dot labeled antibody molecules using multifocal plane microscopy.

Single molecule tracking in three dimensions (3D) in a live cell environment promises to reveal important new insights into cell biological mechanisms. However, classical microscopy techniques suffer from poor depth discrimination which severely limits single molecule tracking in 3D with high temporal and spatial resolution. We introduced a novel imaging modality, multifocal plane microscopy (MUM) for the study of subcellular dynamics in 3D. We have shown that MUM provides a powerful approach with which single molecules can be tracked in 3D in live cells. MUM allows for the simultaneous imaging at different focal planes, thereby ensuring that trajectories can be imaged continuously at high temporal resolution. A critical requirement for 3D single molecule tracking as well as localization based 3D super-resolution imaging is high 3D localization accuracy. MUM overcomes the depth discrimination problem of classical microscopy based approaches and supports high accuracy 3D localization of singe molecule/particles. In this way, MUM opens the way for high precision 3D single molecule tracking and 3D super-resolution imaging within a live cell environment. We have used MUM to reveal complex intracellular pathways that could not be imaged with classical approaches. In particular we have tracked quantum dot labeled antibody molecules in the exo/endocytic pathway from the cell interior to the plasma membrane at the single molecule level. Here, we present a brief review of these results.

Improved single particle localization accuracy with dual objective multifocal plane microscopy.

In single particle imaging applications, the number of photons detected from the fluorescent label plays a crucial role in the quantitative analysis of the acquired data. For example, in tracking experiments the localization accuracy of the labeled entity can be improved by collecting more photons from the labeled entity. Here, we report the development of dual objective multifocal plane microscopy (dMUM) for single particle studies. The new microscope configuration uses two opposing objective lenses, where one of the objectives is in an inverted position and the other objective is in an upright position. We show that dMUM has a higher photon collection efficiency when compared to standard microscopes. We demonstrate that fluorescent labels can be localized with better accuracy in 2D and 3D when imaged through dMUM than when imaged through a standard microscope. Analytical tools are introduced to estimate the nanoprobe location from dMUM images and to characterize the accuracy with which they can be determined.

Dual objective fluorescence microscopy for single molecule imaging applications.

Fluorescence microscopy is an invaluable tool for studying biological processes in cells. In the recent past there has been significant interest in imaging cellular processes at the single molecule level. Single molecule experiments remove ensemble averaging effects and provide information that is typically not accessible through bulk experiments. One of the major requirements in single molecule imaging applications is that a sufficient number of photons be detected from the single molecule. This is not only important for the visual identification of single molecules, but also plays a crucial role in the quantitative analysis of the acquired data. Here, we demonstrate the use of a dual objective imaging configuration for single molecule studies. The configuration uses two opposing objective lenses, where one of the objectives is in an inverted position and the other objective is in an upright position. The use of opposing objective lenses has been previously demonstrated in 4pi confocal microscopy and I5M to achieve high axial resolution when compared to confocal/widefield microscopes. Here we demonstrate that the dual objective imaging configuration provides higher photon collection efficiency when compared to a regular microscope for a given illumination condition. As a result, single molecules can be localized with better accuracy when imaged through opposing objective lenses than when imaged through a regular optical microscope. Analytical tools are introduced to estimate the 2D location of single molecules and to characterize the accuracy with which they can be determined.

High accuracy 3D quantum dot tracking with multifocal plane microscopy for the study of fast intracellular dynamics in live cells.

Single particle tracking in three dimensions in a live cell environment holds the promise of revealing important new biological insights. However, conventional microscopy-based imaging techniques are not well suited for fast three-dimensional (3D) tracking of single particles in cells. Previously we developed an imaging modality multifocal plane microscopy (MUM) to image fast intracellular dynamics in three dimensions in live cells. Here, we introduce an algorithm, the MUM localization algorithm (MUMLA), to determine the 3D position of a point source that is imaged using MUM. We validate MUMLA through simulated and experimental data and show that the 3D position of quantum dots can be determined over a wide spatial range. We demonstrate that MUMLA indeed provides the best possible accuracy with which the 3D position can be determined. Our analysis shows that MUM overcomes the poor depth discrimination of the conventional microscope, and thereby paves the way for high accuracy tracking of nanoparticles in a live cell environment. Here, using MUM and MUMLA we report for the first time the full 3D trajectories of QD-labeled antibody molecules undergoing endocytosis in live cells from the plasma membrane to the sorting endosome deep inside the cell.

Localizing single molecules in three dimensions - a brief review.

Single molecule tracking in three dimensions (3D) in a live cell environment holds the promise of revealing important new biological insights. However, conventional microscopy based imaging techniques are not well suited for fast 3D tracking of single molecules in cells. Previously we developed an imaging modality multifocal plane microscopy (MUM) to image fast intracellular dynamics in 3D in live cells. Recently, we have reported an algorithm, the MUM localization algorithm (MUMLA), for the 3D localization of point sources that are imaged using MUM. Here, we present a review of our results on MUM and MUMLA. We have validated MUMLA through simulated and experimental data and have shown that the 3D-position of quantum dots (QDs) can be determined with high spatial accuracy over a wide spatial range. We have calculated the Cramer-Rao lower bound for the problem of determining the 3D location of point sources from MUM and from conventional microscopes. Our analyses shows that MUM overcomes the poor depth discrimination of the conventional microscope, and thereby paves the way for high accuracy tracking of nanoparticles in a live cell environment. We have also shown that the performance of MUMLA comes consistently close to the Cramer-Rao lower bound.

Elucidation of intracellular recycling pathways leading to exocytosis of the Fc receptor, FcRn, by using multifocal plane microscopy.

The intracellular events on the recycling pathway that lead from sorting endosomes to exocytosis at the plasma membrane are central to cellular function. However, despite intensive study, these processes are poorly characterized in spatial and dynamic terms. The primary reason for this is that, to date, it has not been possible to visualize rapidly moving intracellular compartments in three dimensions in cells. Here, we use a recently developed imaging setup in which multiple planes can be simultaneously imaged within the cell in conjunction with visualization of the plasma membrane plane by using total internal reflection fluorescence microscopy. This has allowed us to track and characterize intracellular events on the recycling pathway that lead to exocytosis of the MHC Class I-related receptor, FcRn. We observe both direct delivery of tubular and vesicular transport containers (TCs) from sorting endosomes to exocytic sites at the plasma membrane, and indirect pathways in which TCs that are not in proximity to sorting endosomes undergo exocytosis. TCs can also interact with different sorting endosomes before exocytosis. Our data provide insight into the intracellular events that precede exocytic fusion.

Simultaneous imaging of different focal planes in fluorescence microscopy for the study of cellular dynamics in three dimensions.

The imaging of cellular dynamics in three dimensions using a standard microscope is severely limited due to the fact that only one focal plane can be imaged at a given point in time. Here we present a modification of the classical microscope design with which two or more focal planes can be imaged simultaneously. This is achieved by a modification of the emission pathway of a standard microscope. The efficacy of the design is shown by imaging bead samples and an FcRn-green fluorescent protein expressing tubule that leaves a sorting endosome and subsequently exocytoses at the plasma membrane.