Optical nanoscopy allows to study biological and functional processes of sub-cellular organelles. In structured
illumination microscopy (SIM) the sample is illuminated with a grid-like interference pattern to encode higher spatial
frequency information into observable Moiré patterns. By acquiring multiple images and a computation trick a superresolved
image is obtained. SIM provides resolution enhancement of 2X in each axis as compared to conventional
microscopes. For a visible light, SIM provides an optical resolution of 100 nm. The challenges associated with optical
nanoscopy of a living cell are photo-toxicity, special dye requirements and artifacts due to cell movement. SIM works
with conventional dyes and is a wide-field technique making it suitable for imaging living cells. In this work, we will
discuss the opportunities and challenges of imaging living cells using SIM. Two applications of optical nanoscopy of
living cells will be discussed; a) imaging of mitochondria in a keratinocyte cell and
Optical microscopy based on fluorescence has emerged as a vital tool in modern bio-medical imaging and diagnosis.
Super-resolution bio-imaging allows gathering information from sub-cellular organelles. In structured illumination
microscopy (SIM) the sample is illuminated with a grid-like interference patterns to encode higher spatial frequencies
information into observable images (Moiré fringes). A super-resolved image is then decoded using computational trick.
In this work, we used SIM to acquired super-resolved optical images of mitochondria from a live keratinocyte cell (see
Fig 1). SIM provides resolution enhancement of 2X in each axis and contrast enhancement of 8X on a projected image.
Time-lapsed imaging was used to study the dynamics of mitochondria in a live cell.
Autophagy is an intracellular recycling mechanism that helps cells to survive against environmental stress and nutritional
starvation. We have recently shown that prostate cancers undergo metabolic stress and caspase-independent cell death
following exposure to arginine deiminase (ADI, an enzyme that degrades arginine in tissue). The aims of our current
investigation into the application of ADI as a novel cancer therapy are to identify the components mediating tumor cell
death, and to determine the role of autophagy (stimulated by ADI and/or rapamycin) on cell death. Using advanced
fluorescence microscopy techniques including 3D deconvolution and superresolution structured-illumination microscopy
(SIM), we show that prostate tumor cells that are killed after exposure to ADI for extended periods, exhibit a
morphology that is distinct from caspase-dependent apoptosis; and that autophagosomes forming as a result of ADI
stimulation contain DAPI-stained nuclear material. Fluorescence imaging (as well as cryo-electron microscopy) show a
breakdown of both the inner and outer nuclear membranes at the interface between the cell nucleus and aggregated
autophagolysosomes. Finally, the addition of N-acetyl cysteine (or NAC, a scavenger for reactive oxygen species)
effectively abolishes the appearance of autophagolysosomes containing nuclear material. We hope to continue this
research to understand the processes that govern the survival or death of these tumor cells, in order to develop methods
to improve the efficacy of cancer pharmacotherapy.
In response to a broad-based need for point-of-care multiplex diagnostic capability, we have developed a novel hybrid platform to analyze optically encoded microspheres arranged on a 2-dimensional planar array. The microspheres which we have initially selected are developed by Luminex Inc. as substrates for sandwich-type fluorescent immunoassays and are typically used in conjunction with a customized flow analyzer. CCD-based optics are the essential feature which enables the development of a rugged diagnostic instrument which can be scaled for point-of-care applications. We have characterized the Multiplex Immunoassay Diagnostic System (MIDS) using a benchtop prototype built around a conventional 12-bit CCD. This system is capable of resolving up to 6 discrete classes of fluorescent microbeads, and measuring their corresponding reporter signal. The MIDS sensitivity to the phycoerythrin (PE) reporter compared favorably to that of the reference Luminex flow system, and is capable of identifying viral, bacterial, and protein simulants in laboratory samples, at concentrations less than 1μg/ml. The ability to
resolve small differences in the average PE fluorescence is a direct function of CCD performance, and may be a necessary trade-off for developing a portable and economical detection system. However, we are
confident that the MIDS platform can easily be scaled to meet the nominal requirements of any given point-of-care or screening application, and furthermore provide much-needed diagnostic functionality in this particular environment.
Proceedings Volume Editor (1)
This will count as one of your downloads.
You will have access to both the presentation and article (if available).
Access to the requested content is limited to institutions that have purchased or subscribe to SPIE eBooks.
You are receiving this notice because your organization may not have SPIE eBooks access.*
*Shibboleth/Open Athens users─please
sign in
to access your institution's subscriptions.
To obtain this item, you may purchase the complete book in print or electronic format on
SPIE.org.
INSTITUTIONAL Select your institution to access the SPIE Digital Library.
PERSONAL Sign in with your SPIE account to access your personal subscriptions or to use specific features such as save to my library, sign up for alerts, save searches, etc.