Light microscopy is a widely used tool in biomedical research. Fluorescence microscopy concentrates on quantitative and qualitative measurements of the fluorescent light emitted from the specimen under study. This is generally done using fluorescent molecules that can be tagged to antibodies, giving specific information about the micro-environment of the sample, i.e. oxygen concentration (tissue hypoxia), and/or to visualize specific structures, such as, tissue morphology (H & E), and blood vessel location (CD31). Biological applications of fluorescence microscopy such as imaging cut and stained tissue/tumour sections use specimens that 'overfill' the field of view of standard microscope objectives. An average tumour in these studies is 5-10 mm wide, while microscope objectives range in their field of view from -1 mm down to a few hundred microns, with smaller fields as magnification power increases. This can pose some difficulties for studies that look at the expression of a parameter across the entire specimen.
We report results from a proof-of-principle study investigating a technique for high-resolution imaging of large fields of view (FOV). This is achieved through structured illumination of the sample from a laterally replicated spatial light modulator (SLM). By incorporating the SLM into the illumination path of an otherwise conventional microscopy imaging system, we can perform the sampling by using our illumination source instead of our areal detector (camera). The increased resolution is achieved through anti-binning or splitting of the charge-coupled device (CCD) pixels, and the extended FOV is obtained by a lateral replication technique applied to the whole illumination field. With anti-binning, we effectively exceed the sampling resolution limit set by the Nyquist theorem. Also, our lateral replication technique enables us to maintain the same FOV for the increased resolution without the need for adaptive optics or highly corrected lenses far from the optical axis. The two techniques of resolution enhancement and lateral replication of the illumination field could be employed independently, hence offering increased versatility and adaptability for specialized imaging applications. Different imaging modes can be accessed digitally, without the need to change objectives, stitch together individual frames, or move the sample. The resulting imaging modality of this system is quasi-confocal.
The use of digital fluorescence confocal microscopy in biological sciences has grown in recent decades due to the versatility of fluorescence imaging. The ability to selectively label specific morphological features, genetic mutations and/or chemical micro-environmental changes with discreet fluorescent labels allows a better understanding of the complex systems that regulate cellular processes. Specimens can range in size from single cells to tissue sections and tissue arrays, which can occupy the entire surface of a microscope slide (25mm x 70mm). Using a confocal scanning laser MACROscope, a wide-area confocal imaging system (Biomedical Photometrics Inc.), it is possible to image these large specimens at high resolution, without the need to tile many small microscope fields. A hyperspectral imaging (HSI) mode has been added to the MACROscope system to assess the use of HSI in the removal/separation of tissue autofluorescence from digital images of fluorescently-labeled paraffin-embedded, formalin-fixed tissue sections. In pathology and immunohistochemistry applications this autofluorescence can hinder, or even prevent, detection of the applied fluorescent label(s). In the present study, fluorescence emission from the specimen was sampled at ~7 nm bandwidths across 32 channels, amounting to viewing ~220 nm of the visible spectrum as a hyperspectral data cube. The data cube was then processed to remove the contributions from autofluorescence, leaving only the signal from the fluorophore(s) of interest. Comparisons are drawn from HSI obtained with a commercial hyperspectral confocal microscope (Zeiss LSM 510 META) employing image tiling. The initial results demonstrate the ability to spectrally unmix the tissue autofluorescence in large tissue sections.
Recent advances in imaging technology have contributed greatly to biological science. Confocal fluorescence microscopy (CFM) facilitates high-contrast 2D and 3D images of biological samples such as living cells, and frozen or fixed tissue sections. However, to date, imaging with existing confocal microscopes has been limited by practicality, especially when samples are large and overfill the field of view (FOV) of typical microscope objectives (e.g., ~10 mm2 tissue section). In this case, image-tiling must be employed to cover the entire specimen. This can be time consuming and cause artifacts in the composite image. The MACROscope® system (Biomedical Photometrics Inc, Waterloo, Canada), is a confocal device with a 2x7 cm2 FOV, and is ideal for imaging large tissue sections in a single frame. The system used in this work is a prototype capable of simultaneous acquisition from two detection channels. Reflected light (RL), transmitted light (TL) and differential phase contrast (DPC) images of whole cut mouse tumor xenografts were collected with the same system. Preliminary results demonstrate that the MACROscope® can produce high quality images of large tissue samples; comparable in resolution and contrast to those obtained with conventional CFM using low-power (5-10x) objectives, but at increased imaging speeds (>10x), and FOV (>20x). This new device avoids the need for image-tiling and provides simultaneous imaging of multiple tissue-specific fluorescent labels in large biological samples with high resolution and contrast; thereby allowing time- and cost-efficient high-throughput screening of immunohistopathological samples. This device may also serve in the imaging of high-throughput DNA and tissue arrays.