Structured illumination microscopy using random intensity incoherent reflectance

Abstract

Depth information is resolved from thick specimens using a modification of structured illumination. By projecting a random projection pattern with varied spatial frequencies that is rotated while capturing images, sectioning can be performed using an incoherent light source in reflectance only. This provides a low-cost solution to obtaining information similar to that produced in confocal microscopy and other methods of structured illumination, without the requirement of complex or elaborate equipment, coherent light sources, or fluorescence. The broad line width of the light emitting diode minimizes artifacts associated with speckle from the laser while also increasing the safety of the instrument. Single diffusers and cascaded diffusers are compared to provide the most efficient method for sectioning at depth. By using reflectance only, in vivo images are produced on a human subject, generating high-contrast images and providing depth information about subsurface objects.

Assessing resolution in super-resolution imaging

“…Typically this is done by illuminating with stripes and phase-shifting the stripes over a complete cycle [12,21]. Many other schemes are possible, including random patterns [22] or unknown patterns [23]. The data is then processed to extract super-resolution information in the direction of the stripes.…”

Techniques for fluorescence detection of protoporphyrin IX in skin cancers associated with photodynamic therapy

“…They achieved a two-fold increase in resolution compared with standard wide-field microscopy and higher resolving power than confocal and multiphoton microscopy. Using complementary techniques like random intensity illumination and rotating view acquisition in combination with SIM, better depth information and increased reconstruction quality has been achieved in human and animal subjects [69, 70]. …”

High-resolution fluorescence imaging via pattern-illuminated Fourier ptychography

“…We reiterate that, the use of non-uniform intensity patterns for sample illumination is not a new idea. It has been demonstrated in various imaging settings for improving lateral or axial resolution [2][3][4][5][6][7][8][9][11][12][13][14][15][16][17][18][19]. However, the use of the FP framework for recovering a highresolution fluorescence image is new and may provide an alternative solution for the existing SIM and speckle illuminating platforms.…”

“…We note that, the strategy of using non-uniform illumination for improving lateral or axial resolution is not new [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21]. However, the use of the FP framework [22][23][24][25][26][27][28] to recover a high-resolution fluorescence image is new and may provide an alternative solution to existing SIM and speckle illuminating platforms.…”

“…Similar to SIM and other speckle illumination schemes [2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19][20][21], the reported approach uses non-uniform intensity patterns for sample illumination and acquires the corresponding diffraction-limited intensity images. Based on the acquired images, a novel Fourier ptychographic recovery algorithm is used to reconstruct the high-resolution sample image in an iterative manner.…”