. 2023 May 25;6:559. doi: 10.1038/s42003-023-04934-8

Table 1.

Super biotargets for labelfree optical microscopy.

Structure		Function	Compatibility with labelfree optical microscopes	Integrability into biomedical research
Extra cellular	Collagen^{6, 55,84,144–147}	Most abundant protein, present in connective tissues, main component of the extracellular matrix, organ mechanical integrity, contains the amino acid sequence for cell-matrix adhesion, several isotypes found in the body abnormality can occur in almost any organ with solid connective tissue	Modalities: QPM, OCT (light scattering); polarization sensitive-OCT (birefringent); SHG (fibrillar structure); AF (autofluorescent); optical superresolution (AF, SHG etc.); Raman/FTIR microspectroscopy (triple helix protein, side-chain modifications chemistry, crosslinking chemistry); BM (strong Brillouin shift), OCE (optical response to deformation) Challenges: very dense - inhibiting light penetration, highly scattering - prone to noise	Usable formats: isolated commercial grade collagen from different animal origins, tissue sections (formalin-fixed, cryopreserved), 3D collagen matrix for in vitro cell culture, 3D engineered tissue, in vivo/ex vivo tissues Spatial correlatability: staining methods (e.g., van Gieson’s, Mason trichrome, Mallory trichrome, Movat’s pentachrome); immunolabelled for all or specific isotypes. Application: organ fibrosis, development, tissue repair, musculoskeletal disorders, cancers
Blood/ vasculature^{31, 40,148–154}	Includes blood cells and vessels, transport respiratory gases, nutrition, hormones, water, and waste material across the body, an important part of the immune system, wound repair abnormality can affect all organs	Modalities: QPM (RBC, tissue sections); OCT (tissues); AF (RBC), optical superresolution based on AF; PAM (hemoglobin pigment) Raman/FTIR microspectroscopy (chemical changes in hemoglobin), BM (vessel lining, blood cells), OCE (vessel lining stiffness response) Challenges: present deep into the tissue, restricting in situ imaging in larger organs and animals	Usable formats: blood smears, tissue sections, engineered in vitro blood vessels Spatial correlatability: blood cell staining methods, immunolabelling for endothelium (cells and lining matrix) Application: vasculogenesis, angiogenesis in development and cancer, anemia, leukemia
Intra cellular	Cytoskeleton^{45, 56–58,131,155}	Includes actin, myosin, microtubules, and intermediate filaments; cell shape and structure; cell motility, cell growth and differentiation, both cell-cell and cell-matrix interaction, cell division, mechano-sensation from membrane to nucleus abnormality can affect cell structure and function	Modalities: QPM, superresolution (QPM based), SHG (fibrillar structure of actomyosin, microtubules), Raman/FTIR microspectroscopy (branching, crosslinking), BM (good Brillouin shift), Challenges: difficult to differentiate from other intracellular structures in labelfree methods	Usable formats: commercially available isolated, in vitro cell culture, tissue sections. Spatial correlatability: direct and indirect fluorescence labeling application: cell biology, fibrosis, cancer, motility, growth, cell stiffness.
Nucleus^{32, 81,156–158}	Includes nuclear membrane, nucleoplasm, nucleolus, and chromatin material, in all adult eukaryotic cells except RBCs, multiple nuclei in some cells like muscles, cell division, genetic information, repair, replication, transcription abnormality can affect cell multiplication and genetic propagation	Modalities: QPM, AF, AF superresolution, Raman/FTIR microspectroscopy, BM challenges: no major challenges	Usable formats: in vitro cells, 3D cell-laden engineered tissues, tissue sections Spatial correlatability: nuclear stains (whole nucleus), chromatin stains (e.g., Giemsa), membrane stains (nuclear membrane) Application: genetic diseases, cancers, cell biology, development, differentiation

Structure

Function

Compatibility with labelfree optical microscopes

Integrability into biomedical research

Extra cellular

Collagen^{6, 55,84,144–147}

Most abundant protein, present in connective tissues, main component of the extracellular matrix, organ mechanical integrity, contains the amino acid sequence for cell-matrix adhesion, several isotypes found in the body

abnormality can occur in almost any organ with solid connective tissue

Modalities: QPM, OCT (light scattering); polarization sensitive-OCT (birefringent); SHG (fibrillar structure); AF (autofluorescent); optical superresolution (AF, SHG etc.); Raman/FTIR microspectroscopy (triple helix protein, side-chain modifications chemistry, crosslinking chemistry); BM (strong Brillouin shift), OCE (optical response to deformation)

Challenges: very dense - inhibiting light penetration, highly scattering - prone to noise

Usable formats: isolated commercial grade collagen from different animal origins, tissue sections (formalin-fixed, cryopreserved), 3D collagen matrix for in vitro cell culture, 3D engineered tissue, in vivo/ex vivo tissues

Spatial correlatability: staining methods (e.g., van Gieson’s, Mason trichrome, Mallory trichrome, Movat’s pentachrome); immunolabelled for all or specific isotypes.

Application: organ fibrosis, development, tissue repair, musculoskeletal disorders, cancers

Blood/ vasculature^{31, 40,148–154}

Includes blood cells and vessels, transport respiratory gases, nutrition, hormones, water, and waste material across the body, an important part of the immune system, wound repair

abnormality can affect all organs

Modalities: QPM (RBC, tissue sections); OCT (tissues); AF (RBC), optical superresolution based on AF; PAM (hemoglobin pigment) Raman/FTIR microspectroscopy (chemical changes in hemoglobin), BM (vessel lining, blood cells), OCE (vessel lining stiffness response)

Challenges: present deep into the tissue, restricting in situ imaging in larger organs and animals

Usable formats: blood smears, tissue sections, engineered in vitro blood vessels

Spatial correlatability: blood cell staining methods, immunolabelling for endothelium (cells and lining matrix)

Application: vasculogenesis, angiogenesis in development and cancer, anemia, leukemia

Intra cellular

Cytoskeleton^{45, 56–58,131,155}

Includes actin, myosin, microtubules, and intermediate filaments; cell shape and structure; cell motility, cell growth and differentiation, both cell-cell and cell-matrix interaction, cell division, mechano-sensation from membrane to nucleus

abnormality can affect cell structure and function

Modalities: QPM, superresolution (QPM based), SHG (fibrillar structure of actomyosin, microtubules), Raman/FTIR microspectroscopy (branching, crosslinking), BM (good Brillouin shift),

Challenges: difficult to differentiate from other intracellular structures in labelfree methods

Usable formats: commercially available isolated, in vitro cell culture, tissue sections.

Spatial correlatability: direct and indirect fluorescence labeling

application: cell biology, fibrosis, cancer, motility, growth, cell stiffness.

Nucleus^{32, 81,156–158}

Includes nuclear membrane, nucleoplasm, nucleolus, and chromatin material, in all adult eukaryotic cells except RBCs, multiple nuclei in some cells like muscles, cell division, genetic information, repair, replication, transcription

abnormality can affect cell multiplication and genetic propagation

Modalities: QPM, AF, AF superresolution, Raman/FTIR microspectroscopy, BM

challenges: no major challenges

Usable formats: in vitro cells, 3D cell-laden engineered tissues, tissue sections

Spatial correlatability: nuclear stains (whole nucleus), chromatin stains (e.g., Giemsa), membrane stains (nuclear membrane)

Application: genetic diseases, cancers, cell biology, development, differentiation

Some biological targets have tremendous potential due to their versatility and amenability to labelfree microscopy as well as their practical impact on biological and medical research. Here are enlisted some of the super biotargets and their potential for labelfree microscopy.