Biological research
,H1: EXPERIMENTAL DESIGN ................................................................................................................. 1
1.1 SCIENCE?.................................................................................................................................................. 1
1.2 PRINCIPLES OF SCIENCE ............................................................................................................................ 1
1.3 TYPES OF SCIENTIFIC RESEARCH ............................................................................................................... 2
1.4 SAMPLING ................................................................................................................................................. 3
1.5 WHAT IS “GOOD” EXPERIMENTAL DESIGN ?................................................................................................ 4
1.6 STEPS TO DEVELOP EXPERIMENTAL DESIGN .............................................................................................. 4
H2: RULES OF SCIENCE AND CRITICAL THINKING ........................................................................... 7
2.1 THE EMPIRICAL CYCLE ............................................................................................................................... 7
2.2 THE SIX RULES OF SCIENCE ....................................................................................................................... 8
2.3 BAD SCIENCE – EXAMPLE EXERCISE .......................................................................................................... 9
H3: BIOLOGICAL MODELS ..................................................................................................................11
3.1 INTRODUCTION AND HISTORY OF BIOLOGICAL MODELS ........................................................................... 11
3.2 CRITERIA TO BE A GOOD BIOLOGICAL MODEL .......................................................................................... 12
3.3 THE MAIN BIOLOGICAL MODEL ORGANISMS ............................................................................................. 13
H4: LIGHT MICROSCOPY ....................................................................................................................24
4.1 INTRODUCTION ........................................................................................................................................ 24
4.2 BRIGHTFIELD MICROSCOPY ..................................................................................................................... 27
4.3 DARKFIELD MICROSCOPY ........................................................................................................................ 28
4.4 PHASE CONTRAST MICROSCOPY ............................................................................................................. 29
4.5 DIFFERENTIAL INTERFERENCE CONTRAST MICROSCOPY (NOMARSKI) ..................................................... 30
4.6 FLUORESCENCE MICROSCOPY ................................................................................................................ 31
4.7 CONFOCAL MICROSCOPY........................................................................................................................ 32
H5: ELECTRONIC MICROSCOPY ........................................................................................................33
5.2 TRANSMISSION ELECTRON MICROSCOPY (TEM) .................................................................................... 33
5.3 SCANNING ELECTRON MICROSCOPY (SEM)............................................................................................ 36
5.4 SCANNING TUNNELING MICROSCOPY (STM) .......................................................................................... 39
5.5 ATOMIC FORCE MICROSCOPY (AFM) ...................................................................................................... 39
H6: SPECTROSCOPY ...........................................................................................................................43
6.1 ABSORPTION SPECTROSCOPY ................................................................................................................ 43
6.2 FLUORESCENCE SPECTROSCOPY ............................................................................................................ 47
6.3 INFRARED SPECTROSCOPY ...................................................................................................................... 50
6.4 RAMAN SPECTROSCOPY.......................................................................................................................... 52
6.7 NMR SPECTROSCOPY ............................................................................................................................. 55
H7: ELECTROPHORESIS ......................................................................................................................57
7.1 BACKGROUND ......................................................................................................................................... 57
7.2 DENATURING GRADIENT GEL ELECTROPHORESIS (DGGE) ...................................................................... 58
7.3 TEMPERATURE GRADIENT GEL ELECTROPHORESIS (TGGE) .................................................................... 60
7.4 PULSE-FIELD ELECTROPHORESIS (PFGE) ............................................................................................... 61
7.5 CAPILLARY ELECTROPHORESIS (CE) ....................................................................................................... 62
7.6 ELECTROPHORESIS OF PROTEINS ............................................................................................................ 63
7.7 OVERVIEW ELECTROPHORESIS ................................................................................................................ 64
,H8: CHROMATOGRAPHY ....................................................................................................................65
8.1 INTRODUCTION AND GENERAL PRINCIPLES .............................................................................................. 65
8.2 COLUMN CHROMATOGRAPHY .................................................................................................................. 66
8.3 PAPER AND THIN LAYER CHROMATOGRAPHY ........................................................................................... 68
8.4 HIGH-PRESSURE LIQUID CHROMATOGRAPHY (HPLC) ............................................................................. 70
8.5 GAS CHROMATOGRAPHY ......................................................................................................................... 71
8.6 ION EXCHANGE CHROMATOGRAPHY ........................................................................................................ 73
8.7 LIQUID CHROMATOGRAPHY-MASS SPECTROSCOPY (LC/MS) ................................................................. 74
H9: PROTEOMICS .................................................................................................................................75
9.1 INTRODUCTION ........................................................................................................................................ 75
9.2 STRATEGIES FOR PROTEIN SEPARATION .................................................................................................. 77
9.3 GEL-BASED SEPARATION TECHNIQUE: 2DGE .......................................................................................... 77
9.4 LIQUID-CHROMATOGRAPHY BASED SEPARATION TECHNIQUES ................................................................ 79
9.5 STRATEGIES FOR IDENTIFYING PROTEINS ................................................................................................ 81
H10: REMOTE SENSING .......................................................................................................................84
10.1 WHAT IS REMOTE SENSING .................................................................................................................... 84
10.2 REMOTE SENSING: PLATFORMS AND SPATIAL RESOLUTION ................................................................... 84
10.3 DIFFERENT KINDS OF REMOTE SENSING ................................................................................................ 86
10.4 ADVANTAGES AND DISADVANTAGES OF REMOTE SENSING .................................................................... 87
10.5 BIOLOGICAL APPLICATIONS ................................................................................................................... 87
10.6 EXAMPLE EXERCISES............................................................................................................................. 88
H11: NEXT GENERATION SEQUENCING............................................................................................90
11.1 INTRODUCTION...................................................................................................................................... 90
11.2 NGS TECHNIQUES ................................................................................................................................ 92
11.3 ILLUMINA ............................................................................................................................................... 93
11.4 IONTORRENT ......................................................................................................................................... 93
11.5 PACBIO ................................................................................................................................................. 93
11.6 OXFORD NANOPORE ............................................................................................................................. 94
11.7 APPLICATIONS OF NGS ........................................................................................................................ 94
11.8 OVERVIEW NGS .................................................................................................................................... 95
11.9 EXAMPLES OF RESEARCH QUESTIONS AND FITTING NGS ..................................................................... 95
H12: ECOSYSTEM RESEARCH ............................................................................................................97
12.1 INTRODUCTION...................................................................................................................................... 97
12.2 CLIMATE MANIPULATION ........................................................................................................................ 97
12.3 FREE-AIR CO2 ENRICHMENT (FACE)................................................................................................... 100
12.4 FREE-AIR TEMPERATURE INCREASE (FATI) .......................................................................................... 101
12.5 RAIN SIMULATORS, RAIN CURTAINS AND RAINOUT SHELTERS .............................................................. 102
12.6 COMBINATIONS IN THE FIELD ............................................................................................................... 102
12.7 ECOTRONS .......................................................................................................................................... 103
12.8 ENCLOSED AQUATIC SYSTEMS ............................................................................................................ 103
12.9 CONCLUSION ...................................................................................................................................... 103
OVERALL: CHOOSING THE RIGHT TECHNIQUE............................................................................ 104
, H1: Experimental design
1.1 Science?
= a way of knowing things about the natural world, relying on certain principles
= organizes knowledge in the form of testable explanations and predictions about the universe;
relies on empirical evidence obtained through observation, experimentation and logical
reasoning to understand natural phenomena
o Being in a dark room and looking for the black cat using a flashlight
Science is not the only thing to gain information:
• Religion: a set of beliefs, practices and values centred around the worship of a deity or
deities, often involving rituals, moral codes and a worldview that addresses fundamental
questions about existence
o Being in a dark room and believing there is a black cat in the room, without having
ever seen a black cat or seeing a black cat enter the room
• Metaphysics: branch of philosophy that explores fundamental concepts such as
existence, reality, causation, nature of being,.. extending beyond the empirical
observations of the physical world
o Being in a dark room and looking for a black cat that isn’t there
• Philosophy: systematic inquiry into fundamental questions about knowledge, existence,
ethics, reason, mind and language, using critical and logical analysis to seek
understanding and meaning in various aspects of the human experience
o Being in a dark room and looking for a black cat
• Theology: study of the nature of the divine, religious beliefs and the relationship between
humanity and the divine. Often involves interpretation of sacred text and exploration of
religious doctrines and traditions.
o Being in a dark room, looking for a black cat that isn’t there and shouting “I found
it!”
1.2 Principles of science
Science and scientific method rely on fundamental principles:
1. Empiricism: knowledge gained through observation and experimentation
2. Systematic observation: data is collected in an organized and structured manner
3. Reproducibility: experiments and observations should be independently repeatable
4. Falsifiability: scientific theories and hypotheses must be formulated in a way that allows
them to be tested and potentially proven false
5. Objectivity: scientific inquiry should be free from personal bias or preconceived notions
6. Hypothesis testing: formulating clear, testable predictions based on a hypothesis
7. Peer review: evaluation of scientific work by experts in the same field before publication
8. Consistency with existing knowledge: new findings should be consistent with established
scientific principles
9. Openness and transparency: sharing methods, data and findings openly with scientific
community
10. Occam’s razor: when multiple explanations are available is the simplest one often the
most likely
1
,H1: EXPERIMENTAL DESIGN ................................................................................................................. 1
1.1 SCIENCE?.................................................................................................................................................. 1
1.2 PRINCIPLES OF SCIENCE ............................................................................................................................ 1
1.3 TYPES OF SCIENTIFIC RESEARCH ............................................................................................................... 2
1.4 SAMPLING ................................................................................................................................................. 3
1.5 WHAT IS “GOOD” EXPERIMENTAL DESIGN ?................................................................................................ 4
1.6 STEPS TO DEVELOP EXPERIMENTAL DESIGN .............................................................................................. 4
H2: RULES OF SCIENCE AND CRITICAL THINKING ........................................................................... 7
2.1 THE EMPIRICAL CYCLE ............................................................................................................................... 7
2.2 THE SIX RULES OF SCIENCE ....................................................................................................................... 8
2.3 BAD SCIENCE – EXAMPLE EXERCISE .......................................................................................................... 9
H3: BIOLOGICAL MODELS ..................................................................................................................11
3.1 INTRODUCTION AND HISTORY OF BIOLOGICAL MODELS ........................................................................... 11
3.2 CRITERIA TO BE A GOOD BIOLOGICAL MODEL .......................................................................................... 12
3.3 THE MAIN BIOLOGICAL MODEL ORGANISMS ............................................................................................. 13
H4: LIGHT MICROSCOPY ....................................................................................................................24
4.1 INTRODUCTION ........................................................................................................................................ 24
4.2 BRIGHTFIELD MICROSCOPY ..................................................................................................................... 27
4.3 DARKFIELD MICROSCOPY ........................................................................................................................ 28
4.4 PHASE CONTRAST MICROSCOPY ............................................................................................................. 29
4.5 DIFFERENTIAL INTERFERENCE CONTRAST MICROSCOPY (NOMARSKI) ..................................................... 30
4.6 FLUORESCENCE MICROSCOPY ................................................................................................................ 31
4.7 CONFOCAL MICROSCOPY........................................................................................................................ 32
H5: ELECTRONIC MICROSCOPY ........................................................................................................33
5.2 TRANSMISSION ELECTRON MICROSCOPY (TEM) .................................................................................... 33
5.3 SCANNING ELECTRON MICROSCOPY (SEM)............................................................................................ 36
5.4 SCANNING TUNNELING MICROSCOPY (STM) .......................................................................................... 39
5.5 ATOMIC FORCE MICROSCOPY (AFM) ...................................................................................................... 39
H6: SPECTROSCOPY ...........................................................................................................................43
6.1 ABSORPTION SPECTROSCOPY ................................................................................................................ 43
6.2 FLUORESCENCE SPECTROSCOPY ............................................................................................................ 47
6.3 INFRARED SPECTROSCOPY ...................................................................................................................... 50
6.4 RAMAN SPECTROSCOPY.......................................................................................................................... 52
6.7 NMR SPECTROSCOPY ............................................................................................................................. 55
H7: ELECTROPHORESIS ......................................................................................................................57
7.1 BACKGROUND ......................................................................................................................................... 57
7.2 DENATURING GRADIENT GEL ELECTROPHORESIS (DGGE) ...................................................................... 58
7.3 TEMPERATURE GRADIENT GEL ELECTROPHORESIS (TGGE) .................................................................... 60
7.4 PULSE-FIELD ELECTROPHORESIS (PFGE) ............................................................................................... 61
7.5 CAPILLARY ELECTROPHORESIS (CE) ....................................................................................................... 62
7.6 ELECTROPHORESIS OF PROTEINS ............................................................................................................ 63
7.7 OVERVIEW ELECTROPHORESIS ................................................................................................................ 64
,H8: CHROMATOGRAPHY ....................................................................................................................65
8.1 INTRODUCTION AND GENERAL PRINCIPLES .............................................................................................. 65
8.2 COLUMN CHROMATOGRAPHY .................................................................................................................. 66
8.3 PAPER AND THIN LAYER CHROMATOGRAPHY ........................................................................................... 68
8.4 HIGH-PRESSURE LIQUID CHROMATOGRAPHY (HPLC) ............................................................................. 70
8.5 GAS CHROMATOGRAPHY ......................................................................................................................... 71
8.6 ION EXCHANGE CHROMATOGRAPHY ........................................................................................................ 73
8.7 LIQUID CHROMATOGRAPHY-MASS SPECTROSCOPY (LC/MS) ................................................................. 74
H9: PROTEOMICS .................................................................................................................................75
9.1 INTRODUCTION ........................................................................................................................................ 75
9.2 STRATEGIES FOR PROTEIN SEPARATION .................................................................................................. 77
9.3 GEL-BASED SEPARATION TECHNIQUE: 2DGE .......................................................................................... 77
9.4 LIQUID-CHROMATOGRAPHY BASED SEPARATION TECHNIQUES ................................................................ 79
9.5 STRATEGIES FOR IDENTIFYING PROTEINS ................................................................................................ 81
H10: REMOTE SENSING .......................................................................................................................84
10.1 WHAT IS REMOTE SENSING .................................................................................................................... 84
10.2 REMOTE SENSING: PLATFORMS AND SPATIAL RESOLUTION ................................................................... 84
10.3 DIFFERENT KINDS OF REMOTE SENSING ................................................................................................ 86
10.4 ADVANTAGES AND DISADVANTAGES OF REMOTE SENSING .................................................................... 87
10.5 BIOLOGICAL APPLICATIONS ................................................................................................................... 87
10.6 EXAMPLE EXERCISES............................................................................................................................. 88
H11: NEXT GENERATION SEQUENCING............................................................................................90
11.1 INTRODUCTION...................................................................................................................................... 90
11.2 NGS TECHNIQUES ................................................................................................................................ 92
11.3 ILLUMINA ............................................................................................................................................... 93
11.4 IONTORRENT ......................................................................................................................................... 93
11.5 PACBIO ................................................................................................................................................. 93
11.6 OXFORD NANOPORE ............................................................................................................................. 94
11.7 APPLICATIONS OF NGS ........................................................................................................................ 94
11.8 OVERVIEW NGS .................................................................................................................................... 95
11.9 EXAMPLES OF RESEARCH QUESTIONS AND FITTING NGS ..................................................................... 95
H12: ECOSYSTEM RESEARCH ............................................................................................................97
12.1 INTRODUCTION...................................................................................................................................... 97
12.2 CLIMATE MANIPULATION ........................................................................................................................ 97
12.3 FREE-AIR CO2 ENRICHMENT (FACE)................................................................................................... 100
12.4 FREE-AIR TEMPERATURE INCREASE (FATI) .......................................................................................... 101
12.5 RAIN SIMULATORS, RAIN CURTAINS AND RAINOUT SHELTERS .............................................................. 102
12.6 COMBINATIONS IN THE FIELD ............................................................................................................... 102
12.7 ECOTRONS .......................................................................................................................................... 103
12.8 ENCLOSED AQUATIC SYSTEMS ............................................................................................................ 103
12.9 CONCLUSION ...................................................................................................................................... 103
OVERALL: CHOOSING THE RIGHT TECHNIQUE............................................................................ 104
, H1: Experimental design
1.1 Science?
= a way of knowing things about the natural world, relying on certain principles
= organizes knowledge in the form of testable explanations and predictions about the universe;
relies on empirical evidence obtained through observation, experimentation and logical
reasoning to understand natural phenomena
o Being in a dark room and looking for the black cat using a flashlight
Science is not the only thing to gain information:
• Religion: a set of beliefs, practices and values centred around the worship of a deity or
deities, often involving rituals, moral codes and a worldview that addresses fundamental
questions about existence
o Being in a dark room and believing there is a black cat in the room, without having
ever seen a black cat or seeing a black cat enter the room
• Metaphysics: branch of philosophy that explores fundamental concepts such as
existence, reality, causation, nature of being,.. extending beyond the empirical
observations of the physical world
o Being in a dark room and looking for a black cat that isn’t there
• Philosophy: systematic inquiry into fundamental questions about knowledge, existence,
ethics, reason, mind and language, using critical and logical analysis to seek
understanding and meaning in various aspects of the human experience
o Being in a dark room and looking for a black cat
• Theology: study of the nature of the divine, religious beliefs and the relationship between
humanity and the divine. Often involves interpretation of sacred text and exploration of
religious doctrines and traditions.
o Being in a dark room, looking for a black cat that isn’t there and shouting “I found
it!”
1.2 Principles of science
Science and scientific method rely on fundamental principles:
1. Empiricism: knowledge gained through observation and experimentation
2. Systematic observation: data is collected in an organized and structured manner
3. Reproducibility: experiments and observations should be independently repeatable
4. Falsifiability: scientific theories and hypotheses must be formulated in a way that allows
them to be tested and potentially proven false
5. Objectivity: scientific inquiry should be free from personal bias or preconceived notions
6. Hypothesis testing: formulating clear, testable predictions based on a hypothesis
7. Peer review: evaluation of scientific work by experts in the same field before publication
8. Consistency with existing knowledge: new findings should be consistent with established
scientific principles
9. Openness and transparency: sharing methods, data and findings openly with scientific
community
10. Occam’s razor: when multiple explanations are available is the simplest one often the
most likely
1
