Multispectral Imaging Microscope with Millisecond Time Resolution

Abstract

A new multispectral imaging microscope with micrometer spatial resolution and millisecond temporal resolution has been developed. The imaging microscope is based on the use of an acousto-optic tunable filter (AOTF) for spectral tuning and a progressive scan camera capable of snapshot operation for recording. It can operate in two modes: images are recorded as a function of time or wavelength. When operated as a function of time, the microscope is configured so that as many images as possible are recorded, grabbed, and stored per one wavelength. Upon completion, the AOTF is scanned to a new wavelength, and a new set of images are recorded. Up to 33 images/ second (i.e., 30 ms/image) can be recorded in this mode. In the other configuration, the recording wavelength is rapidly scanned (by means of the AOTF) and only one image is rapidly recorded, grabbed, and stored for each wavelength. Because additional time is needed to scan the AOTF, the maximum number of images can be grabbed in this case is 16 frames/s. Preliminary applications of the imaging microscope include measurements of photoinduced changes of a single unit cell in temperature-sensitive cholesteric liquid crystals as a function of time and wavelength. The changes were found to be varied with time and wavelength. Interestingly, the photoinduced changes of unit cells in the liquid crystal are not the same but different from cell to cell. This imaging microscope is particularly useful for measurements of small-size samples that undergo rapid chemical or biochemical reactions, e.g., activities of a single biological cell.

Pulmonary hypertension

“…Near-infrared (NIR) multispectral imaging technique may be the solution to this problem. We have succeeded in developing a novel NIR multispectral imaging (NIR-MSI) instrument that can provide, for the first time, the means to noninvasively, sensitively (single pixel resolution), and rapidly (ms) record not at a single wavelength as in NIR bioimaging instruments but rather on an entire NIR absorption spectrum (as with a Fourier transform [FT]-IR spectrophotometer) and not just one spectrum but rather tens of thousands of spectra at tens of thousands of different locations within a sample during a period as short as a few milliseconds [13][14][15][16][17]. Chemical information derived from these spectra will make it possible to accurately detect and identify any different chemical or biochemical species at many different positions within a sample.…”

“…Because of its advantages, we were able to use the NIR-MSI instrument for studies and measurements that are not possible otherwise. These include the noninvasive and nondestructive authentication of documents (e.g., stock certificates, currency), determination of the chemical inhomogeneity of polymers, kinetics of fast reactions (e.g., curing of epoxy by amine), and detection of combinatorial peptide synthesis [13][14][15][16][17]. It is expected that combined advantages of NIR light (deep tissue penetration) and MSI technique (ability to provide chemical composition and structure at various positions within a sample) enable the NIR-MSI instrument to be uniquely suited for the noninvasive investigation of MCT toxicity in the lung of Sprague-Dawley rats.…”

Determination of Chemical Homogeneity of Fire Retardant Polymeric Nanocomposite Materials by Near-Infrared Multispectral Imaging Microscopy

“…The high sensitivity (single pixel resolution), fast temporal (milliseconds), and high spatial resolution ($mm) of this imaging microscope, make it possible for us to use this multispectral imaging microscope for studies and measurements that, to date, have not been possible using existing techniques (Fischer and Tran 1999a;Fischer and Tran 1999b;Tran 2000;2001;2003;2005). These include photo induced changes of a single unit micron-size cell in temperature-sensitive liquid crystals as a function of time and wavelength, and the determination of molecular state and distribution of fullerenes entrapped in sol-gel samples (Fischer and Tran 1999a;Fischer and Tran 1999b;Khait, Smirnov, and Tran 2001;Tran 2000;2001;2003;2005;Tran, Grishko, and Challa 2008).…”

“…The acousto-optic tunable filter (AOTF) based NIR multispectral imaging microscope used in this work is similar to those used in previous studies (Khait, Smirnov, andTran 2001, Tran 2003;2005;Tran, Grishko, and Challa 2008;Mejac et al 2009) except for the replacement of the focusing microscopic lenses in the microscope with a pair of 0.58 N.A. A 15X reflecting Cassegrains to avoid possible aberrations in the NIR region.…”

“…Recorded multispectral images were analyzed using MatLab. The spatial resolution of this NIR-MSI microscope was determined to be (0.93 AE 03) mm=pixel (Khait, Smirnov, and Tran 2001;Tran 2003;2005;Tran, Grishko, and Challa 2008).…”