The light microscope started to become a serious tool for scientific discovery around 1650, when the Dutchman Anthony van Leeuwenhoek started building and using unique simple devices, which would hold a single glass bead as the magnifying element. It is always amazing, how much he was able to see with such a primitive instrument, as documented in his remarkable drawings. Around the same time Robert Hooke in England also started with a simple magnifier but soon added a second magnification stage for the real first compound microscope. His remarkable discoveries were published in 1 667 in Micrographia Illustrata.
Ligand-binding assays are commonly applied to large numbers of cells in culture; the binding parameters derived from such assays reflect the ensemble average behavior of many cells. Equilibrium binding assays of epidermal growth factor (EGF) binding to the EGF receptor (EGFR) indicate that the EGFR exhibits two affinity states for EGF, one low affinity with Kd about 10 nM and one high affinity with Kd < 1 nM. Bulk binding studies cannot determined if such multiple ligand binding classes are due to cell population heterogeneity or are due to heterogeneity at the individual cell level. Here is described a technique based on single cell imaging of fluorescein-EGF (f-EGF) binding to individual human epidermoid carcinoma A431 cells that demonstrates that both classes of EGFR are found on all A431 cells, that the time course of f-EGF binding to individual cells shows two kinetic on-rates and two off-rates, that cell-to-cell heterogeneity of EGF binding is significant and that ligand binding kinetics vary across an individual cell. Contributions of cell autofluorescence photobleaching and f- EGF photobleaching in the measurement of fluorescent ligand binding are shown to be significant.
Cancer, development, cellular growth and differentiation are governed by gene expression. Recent molecular and cellular advances to visualize and perturb the pathways of transcriptional regulation, nascent RNA processing, and protein trafficking at the single cell level have been developed. More recently, applications utilizing the green fluorescent marker (GFP) from Aequorea victoria have facilitated visualization of these molecular events in a living cell. Specifically, we will describe a novel approach to perturb cellular processes by labeling discrete cellular components of interest with GFP and subsequently altering/ablating them with a laser microbeam.
During the last ten years, microscopy as a scientific tool has reinvented itself. It has changed from a group of principally descriptive methodologies, to a wide range of primary tools and techniques to investigate the molecular organization of organs, tissues and cells. Advances in microscope and camera design, fluorescent dye technologies as well as the advent of inexpensive powerful computers, has made the simultaneous resolution and quantification of multiple concurrent molecular markers for both protein and DNA at a sub-micron resolution a reality. Furthermore, it is possible to probe living cells using a rapidly expanding repertoire of dyes sensitive to changes pH or concentration of specific intracellular ions. An essential extension of these techniques has been the development of methods which allow the distribution of proteins and mRNA to be studied within cells at a molecular resolution by EM. However, concurrent with these developments there has been a more dramatic increase in the level of expertise needed to successfully design experiments, implement these technologies and costs involved in purchasing and maintaining instrumentation. These developments have resulted in a situation such that it is no longer tenable for individual departments or possibly institutions to maintain independent imaging facilities, and centralized resources have become mandatory in order to stay at the cutting edge. This presentation will discuss the problems, pitfalls and advantages of a centralized system, and will focus on implementation, design, data handling, and management of a multi-user facility.
Optical laser trapping microscopy has emerged as a powerful tool not only for the optical manipulation of cells and macromolecules, but also for the study of cellular physiological responses via force transduction and fluorescence imaging. We describe here the most recent results from our laboratory in the use and application of laser trapping microscopy to a variety of studies at the cellular and molecular levels. Fluorescence spectroscopy and imaging have been successfully combined with optical micromanipulation. A single near-infrared laser beam is used for two-photon fluorescence excitation and micromanipulation of trapped biological specimens. Cell viability is observed and monitored with a Nd:YAG laser ((lambda) equals 1064 nm) and an Al:GaAs diode laser ((lambda) equals 809 nm). Traps and conventional fluorescence imaging are also used simultaneously to examine T-cell activation dynamics.
A molecular understanding of biology requires that we establish the in situ functions of the proteins in cellular processes. To address this, we developed chromophore- assisted laser inactivation (CALI) for probing the in vivo function of proteins. CALI inactivates specific proteins in living cells by using non-blocking antibodies conjugated with malachite green (MG) dye. MG absorbs 620 nm laser light (which is not absorbed by cells) to generate short lived free radicals with limited range of oxidative damage (15 angstroms) around the dye. This inactivates the bound protein without significantly affecting its neighbors. CALI has been applied to 40 proteins and achieved specific inactivation in almost all those tested. We have developed micro-CALI which uses a focused laser beam (10 micrometers ) to acutely inactivate specific proteins within cells. We have used this to address the molecular mechanisms of neuronal growth cone motility and has implicated a diversity of proteins (e.g. molecular motors, cytoskeletal, and signaling molecules) in discrete steps of growth cone motility. We hope that micro-CALI will be a useful research tool for addressing dynamic processes in biology and medicine.
Cell damage in UV and NIR laser microscopes by highly focused micromanipulation and fluorescence excitation microbeams has been studied. Damage in erythrocytes, spermatozoa and Chinese hamster ovary cells was detected by monitoring morphology changes, autofluorescence detection, cloning assay, and viability screening. It was found that 364 nm/365 nm UVA radiation induced irreversible cell damage at radiant exposures as low as <10 J/cm2. NIR CW microradiation used in laser tweezers was also able to damage cells via a two-photon excitation process, in particular, when using <800 nm trapping beams. Non- destructive two-photon excitation in femtosecond NIR microscopes is possible within a narrow intensity window. The lower limit is determined by two-photon absorption coefficients and detector efficiency, the higher by intracellular optical breakdown in the extranuclear region. Above certain wavelength-dependent intensity thresholds in femtosecond microscopy, cells were completely destroyed by fragmentation concomitant with plasma generation. The influence of excitation and micromanipulation microbeams should be considered when studying physiology and metabolism of vital cells.
Transport processes are important in biology and medicine. Examples include virus docking and infection, endocytosis of extracellular protein and phagocytosis of antigenic material. Trafficking driven by molecular motors inside a complex 3D environment is a shared common theme. The complex sequence of these events are difficult to resolve with conventional techniques where the action of many cells are asynchronously averaged. Single particle tracking (SPT) was developed by Ghosh and Webb to address this problem and has proven to be a powerful technique in understanding membrane- protein interaction. Since the traditional SPT method uses wide field illumination and area detectors, it is limited to the study of 2D systems. In this presentation, we report the development of a 3D single particle tracking technique using two-photon excitation. Using a real-time feedback system, we can dynamically position the sub-femtoliter two-photon excitation volume to follow the fluorescent particle under transport by maximizing the detected fluorescent intensity. Further, fluorescence spectroscopy can be performed in real time along the particle trajectory to monitor the underlying biochemical signals driving this transport process. The first application of this instrument will focus on the study of antigen endocytosis process of macrophages.
Fluorescence resonance energy transfer (FRET) imaging microscopy is a unique tool to visualize the spatiotemporal dynamics of protein interactions in living cells. Genetic vectors that encode protein fusions with green fluorescent protein (GFP) provide a method for imaging protein localization in living cells. We used FRET to study dimerization of the pituitary-specific transcription factor Pit-1 fused to GFP and BFP. A fusion protein containing GFP separated from BFP by 29 amino acids served as a positive control for FRET. Transcriptional activity of the GFP- and BFP-Pit-1 fusion proteins was demonstrated by their ability to activate the prolactin gene promoter. Using optimized excitation and emission filters, cells expressing the fluorescently-tagged Pit-1 proteins were imaged with a back- thinned, back-illuminated CCD chip that has about 50% quantum efficiency in the blue range. 2D FRET images acquired at the focal plane demonstrated Pit-1 protein associations in the nucleus of living cells. The significance of 2- and 3-D energy transfer imaging from these living cells is discussed.
Laser microbeam irradiation at 820 nm predictably and reproducibly altered morphogenetic patterns in fungal cells. Optical tweezers were highly effective as localized, noninvasive, and largely nondestructive probes under precise spatial and temporal control. In growing hyphae, the position of the Spitzenkorper (a multicomponent complex containing mainly secretory vesicles in the hyphal apex), is correlated with the site of maximum cell expansion during tip growth. The Spitzenkorper was not trapped by the laser, but moved away from the trap, and could be `chased' around the cell by the laser beam. Consequently, the direction of cell elongation was readily changed by moving the Spitzenkorper. When the laser was held steady at the cytoplasmic surface immediately beside the Spitzenkorper, an adventitious branch hypha was initiated on the same side of the hypha, suggesting that unilateral disturbance of vesicle traffic initiated a new lateral Spitzenkorper and hyphal branch near the original hyphal apex. If moving vesicles were trapped by the laser beam and transported to a different area of the cytoplasm near the cell surface, the cell profile bulged where the vesicles were newly concentrated. Variations in the mode of vesicle transfer caused: (1) single and multiple bulges, (2) adventitious branch hyphae, (3) increased cell diameter, and (4) changing directions of hyphal elongation. Thus, laser tweezers emerge as a powerful tool for controlling patterns of cell morphogenesis. The findings strongly support the hypothesis that sites of vesicle concentration and release to the cell surface are important determinants of cell morphogenesis in fungi. This conclusion lends support to the basic premises of a modern mathematical model of hyphal tip growth (the hyphoid/VSC model) but does not in itself provide the information needed for a comprehensive and integrated explanation of the mechanism of cell growth in fungi.
X-ray images of living cells were obtained by using a flash contact x-ray microscope system. The system consists of a high power laser (grass laser or KrF laser) for producing a plasma as an x-ray source, a small vacuum chamber, and a sample holder with a metal target for making plasma. The x- ray images were recorded on an x-ray sensitive layer on a silicon wafer. After chemical development x-ray images on the x-ray sensitive layers were read out by using an atomic force microscope. The performance of the system is demonstrated by presenting x-ray images of unicellular microorganisms, bacteria and skeletal muscle fibers. The intracellular structures were visible in some cases. For laboratory use a flash contact table-top x-ray microscopy is improved using a table-scaled YAG laser. The table-top systems showed good performance comparing to that of the x- ray microscope system with high power laser. These results indicate that the table-top x-ray microscope is a powerful instrumentation for the observation of living cells.
Visual orientation in our 3-dimensional environment just like directional auditory information in the world of sound require 2 spacially separated sensors. The differences in the information our 2 eyes or ears receive allow our brain to add the all important third dimension to the 2-dimensional image, which only one eye can see. Similarly, only one ear would be unable to detect directional information in soundspace.
The crucial step in the diagnostic treatment of skin cancer is the initial examination and detection of any unusual change of a skin lesion. Digital imaging permits the documentation of the size, shape and color of lesions and their later comparison, so the last years its key role as an adjunct to early malignant melanoma diagnosis has arisen. In this work, a novel approach to diagnosis is presented by developing a digital imaging system for capturing and processing the images of individual lesions. It was used a 12 bit CCD camera, connected with a Pentium PC equipped with a Matrox frame grabber and convenient software. Images were collected using a zoom close up lens and a light source, attached to the front of the CCD camera. The format of the images was 640 X 480 pixels with 8 bit color table. The border of the lesion was found using a region growing based algorithm combined with the Gradient Operator. It was calculated the border irregularity and the asymmetry of the lesion, using the compactness formula. There was developed algorithms based on thresholding and region growing to determine the borders of the lesion. Compactness has been calculated as well. The pre-prototype system has been placed at the National Cancer Hospital to support melanoma diagnosis.
In previous studies, we reported 850 nm wavelength optical coherence-gated imaging of biological tissues in vitro and in vivo. The results demonstrated the potential of this technique to show the microstructural differences in living tissues and therefore to non invasively diagnose superficial lesions. However, because of the overwhelming light scattering of most biological tissues, the effective penetration depth of coherence-gated imaging is limited. In order to increase the detection depth and improve imaging contrast, we started the research of fast optical coherence- gated imaging of biological tissues at 1300 nm. The advantages of using long wavelength lie in the following two aspects: (1) Tissue scattering decreases with increasing wavelength; (2) According to our model analysis, coherent interference modulation amplitude also increases with wavelength. The current imaging system can acquire a high- resolution image in less than 30 seconds. Preliminary mouse brain and skin images shows that at 1300 nm, the imaging contrast and penetration depth are improved in comparison with 850 nm, making it useful for living tissue imaging and tumor diagnostics.
The process by which diseases, particularly neoplastic diseases, are diagnosed by pathologists using microscopic evaluation of tissue has changed little over the last several decades despite the advent of molecular medicine. Cells or tissues, stained with complex and sometimes poorly characterized organic dyes, are examined using a subjective, pattern-matching approach whose accuracy and reproducibility has increasingly been challenged. Furthermore, the ability of pathologists to deliver accurate prognoses using histological and clinical parameters remains limited, leading to both over- and under-treatment. While molecular techniques hold out great promise as diagnostic and prognostic tools, they are currently still largely investigational, and costly. A complementary approach is proposed, whereby more information is obtained by improved analysis of conventionally prepared histological and cytological samples. Using inverse Fourier transform multi- pixel spectroscopy, a new instrument has been developed which can display a complete transmittance or emission spectrum at every pixel of an image, providing much more color information than can be appreciated by eye or by conventional red-green-blue color cameras. Since spectra variations in staining behavior correlate with alterations in subcellular macromolecular composition it seems likely that they may also correlate with diagnosis and clinical behavior for a number of disease states. Examples of how this approach may prove useful in clinical practice are provided.