Spatially modulated illumination microscopy: online visualization of intensity distribution and prediction of nanometer precision of axial distance measurements by computer simulations

TY - JOUR

T1 - Spatially modulated illumination microscopy: online visualization of intensity distribution and prediction of nanometer precision of axial distance measurements by computer simulations

AU - Albrecht, B

AU - Failla, A V

AU - Heintzmann, R

AU - Cremer, Christoph

PY - 2001/7

Y1 - 2001/7

N2 - During the last years, measurements considerably beyond the conventional "Abbe-Limit" of optical resolution in far field light microscopy were realized by several light microscopical approaches. Point spread function (PSF) engineering, spectral precision distance microscopy (SPDM), and related methods were used to demonstrate the feasibility of such measurements. SPDM allows the measurement of position and multiple distances between point-like fluorescent objects of different spectral signatures far below the optical resolution criterion as defined by the full width at half maximum of the PSF. Here, we report a software method to obtain online visualization of light distribution in the lateral and axial direction of any object detected in a spatially modulated illumination (SMI) microscope. This strongly facilitates routine application of SMI microscopy. The software was developed using Microsoft Visual C++ running on Windows NT. Furthermore, some aspects of the theoretical limits of the SPDM method were studied by virtual microscopy. For the case of SMI microscopy the precision of axial distance measurements was studied, taking into account photon statistics and image analysis procedures. The results indicate that even under low fluorescence intensity conditions typical for biological structure research, precise distance measurements in the nanometer range can be determined, and that axial distances in the order of 40 nm are detectable with such precision.

AB - During the last years, measurements considerably beyond the conventional "Abbe-Limit" of optical resolution in far field light microscopy were realized by several light microscopical approaches. Point spread function (PSF) engineering, spectral precision distance microscopy (SPDM), and related methods were used to demonstrate the feasibility of such measurements. SPDM allows the measurement of position and multiple distances between point-like fluorescent objects of different spectral signatures far below the optical resolution criterion as defined by the full width at half maximum of the PSF. Here, we report a software method to obtain online visualization of light distribution in the lateral and axial direction of any object detected in a spatially modulated illumination (SMI) microscope. This strongly facilitates routine application of SMI microscopy. The software was developed using Microsoft Visual C++ running on Windows NT. Furthermore, some aspects of the theoretical limits of the SPDM method were studied by virtual microscopy. For the case of SMI microscopy the precision of axial distance measurements was studied, taking into account photon statistics and image analysis procedures. The results indicate that even under low fluorescence intensity conditions typical for biological structure research, precise distance measurements in the nanometer range can be determined, and that axial distances in the order of 40 nm are detectable with such precision.

KW - Computer Simulation

KW - Forecasting

KW - Image Processing, Computer-Assisted

KW - Lasers

KW - Microscopy, Polarization

KW - Software

KW - User-Computer Interface

KW - Journal Article

KW - Research Support, Non-U.S. Gov't

U2 - 10.1117/1.1383293

DO - 10.1117/1.1383293

M3 - SCORING: Journal article

C2 - 11516319

VL - 6

SP - 292

EP - 299

JO - J BIOMED OPT

JF - J BIOMED OPT

SN - 1083-3668

IS - 3

ER -