ऑप्टिकल स्कैटर दो आयामी Gabor फ़िल्टर के आधार पर माइक्रोस्कोपी

Summary

हम एक अंधेरे क्षेत्र माइक्रोस्कोपी एकल जीवित कोशिकाओं के भीतर subcellular गतिशीलता को मापने Gabor फ़िल्टरिंग – तरह के आधार पर विधि प्रदर्शित करता है. तकनीक organelles की संरचना में mitochondrial विखंडन जैसे परिवर्तन के प्रति संवेदनशील है.

Abstract

We demonstrate a microscopic instrument that can measure subcellular texture arising from organelle morphology and organization within unstained living cells. The proposed instrument extends the sensitivity of label-free optical microscopy to nanoscale changes in organelle size and shape and can be used to accelerate the study of the structure-function relationship pertaining to organelle dynamics underlying fundamental biological processes, such as programmed cell death or cellular differentiation. The microscope can be easily implemented on existing microscopy platforms, and can therefore be disseminated to individual laboratories, where scientists can implement and use the proposed methods with unrestricted access.

The proposed technique is able to characterize subcellular structure by observing the cell through two-dimensional optical Gabor filters. These filters can be tuned to sense with nanoscale (10’s of nm) sensitivity, specific morphological attributes pertaining to the size and orientation of non-spherical subcellular organelles. While based on contrast generated by elastic scattering, the technique does not rely on a detailed inverse scattering model or on Mie theory to extract morphometric measurements. This technique is therefore applicable to non-spherical organelles for which a precise theoretical scatter description is not easily given, and provides distinctive morphometric parameters that can be obtained within unstained living cells to assess their function. The technique is advantageous compared with digital image processing in that it operates directly on the object’s field transform rather than the discretized object’s intensity. It does not rely on high image sampling rates and can therefore be used to rapidly screen morphological activity within hundreds of cells at a time, thus greatly facilitating the study of organelle structure beyond individual organelle segmentation and reconstruction by fluorescence confocal microscopy of highly magnified digital images of limited fields of view.

In this demonstration we show data from a marine diatom to illustrate the methodology. We also show preliminary data collected from living cells to give an idea of how the method may be applied in a relevant biological context.

Protocol

1. कोशिकाओं तैयार हो रही है कोशिकाओं है कि दिन के पहले मढ़वाया गया Mitotracker हरे रंग के साथ mitochondria की प्रतिदीप्ति इमेजिंग के लिए लेबल करने की आवश्यकता है. DMSO में Mitotracker हरे रंग की 4 ° सी फ्रीजर से पहले बनाया है…

Discussion

विधि वस्तु है कि कण आकार या उदाहरण के लिए अभिविन्यास सांकेतिक शब्दों में बदलना हो सकता है की पैदावार morphometric नक्शे के ऊपर वर्णित है. यह जानकारी संरचनात्मक कई मायनों में इस्तेमाल किया जा सकता है:

1. यह एक …

Materials

Material Name	Type	Company	Catalogue Number	Comment
DMEM		Invitrogen		Low glucose DMEM
Liebowitz L15 medium		Invitrogen		Without phenol red
L-glutamine		Invitrogen
Mitotracker Green		Invitrogen
Bovine Brain Extract		Clonetics
Fetal Bovine Serum		Gemini Biosciences
Heparin		Sigma
Staurosporine		Sigma
Dymethylsulfoxide		Sigma
Inverted microscope		Carl Zeiss	Axiovert 200M
DMD		Texas Instruments	TI 0.7 XGA DMD 1100
CCD		Roper Scientific	Cascase 512B	High (16 bit) dynamic range CCD
CCD		Roped Scientific	Coolsnap cf